On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates
用于脉冲追踪成像,猝发脉冲追踪成像和基于纳米显微技术的细胞裂解物检测的融合SNAP病毒蛋白的按需标记   

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Journal of Virology
Jun 2018

 

Abstract

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.

Keywords: Fusion proteins (融合蛋白), Stress granules (应激颗粒), RNPs (RNPs), Site-specific protein labeling (特定位点的蛋白质标记), Chemical labeling (化学标记), Cell biology (细胞生物学), Bioimaging (生物成像), Live cell imaging (活细胞成像), Chikungunya (基孔肯雅热病), CHIKV (CHIKV), Alphavirus (甲病毒), Arbovirus (虫媒病毒), nsP3 (nsP3)

Background

To better understand the role of a viral protein during the infectious life cycle, we have adapted existing strategies that facilitate the determination of intracellular protein location, the assessment of protein dynamics in intact cells, and the continued tracking of the viral protein after lysis of the host cell. The first two protocols build on previously published protocols for on-demand SNAP-labeling and the analysis of protein turnover (Bodor et al., 2012). In previous work, our laboratory has fused a viral protein-of-interest, nonstructural protein 3 (nsP3) of Chikungunya virus (CHIKV), to the SNAP-tag, a modified form of a 20-kDa monomeric DNA repair enzyme (Remenyi et al., 2017 and 2018). During SNAP-labeling, the addition of a synthetic O6-benzylguanine (BG) derivative results in a covalent bond between a reactive cysteine residue in the SNAP-tag and the BG-probe (Keppler et al., 2004a and 2004b).

We have also combined SNAP-labeling with a protocol for inspecting cell lysates via light microscopy, which enables visualization of detergent-resistant protein assemblies. Similar approaches have allowed researchers to detect stable granular assemblies of GFP-tagged stress-granule proteins in cell lysates (Jain et al., 2016; Wheeler et al., 2017). Stress granules are assemblies of RNA and protein (RNPs), which form under conditions of cellular stress (Kedersha et al., 2005). It is now possible to isolate the more stable stress granule core from both yeast and mammalian cells (Jain et al., 2016). SNAP-labeling offers an alternative way of tracking tagged viral proteins that may be present in similar subcellular assemblies. Hence, Protocol 3 may not only be useful to study the biochemical nature of viral proteins but also to track any cellular protein that resides in non-membranous organelles such as RNPs and stress granules. For example, integration of the SNAP-tag into the development of cell lines that produce fluorescently tagged stress granules (Kedersha et al., 2008) could increase experimental flexibility during dynamic and quantitative imaging of these cellular sub-compartments.

Our three protocols also take advantage of recently developed commercial imaging systems for multi-color fluorescence microscopy. We analyze labeled samples in Protocol 1 with live-cell imaging (Figure 1) and thus recommend following general procedures for controlling temperature, reducing phototoxicity, limiting photobleaching, and maintaining cell viability (Frigault et al., 2009). The chosen 2018 Nikon Ti-E2 system allowed us to image a large field-of-view, lessen focus drift with a proprietary Perfect Focus System (PFS), and record multiple positions with the motorized stage. Moreover, illumination with an LED and light exposures not exceeding 1 s allowed for gentler imaging compared to the typical imaging setup of a laser scanning confocal microscope.

For the analysis of labeled protein in Protocols 2 and 3, we chose a confocal imaging setup with proprietary ZEISS Airyscan technology (Figure 1), which is a commercial version of the ‘image scanning microscopy’ approach (Muller and Enderlein, 2010; Sheppard et al., 2013). Airyscan microscopy represents one of the recent innovations in fluorescence super-resolution microscopy, also referred to as nanoscopy (Li et al., 2018). The Airyscan technology improves system resolution with an improved detector design, which features a 32-channel detector array (Huff, 2015). With a new 2D super-resolution mode for Airyscan, this detection approach can now enhance resolution 2-fold while lowering the required fluorescence intensity to obtain high-quality images (Huff et al., 2017). The Airyscan system allowed us to stain our samples with fluorescent dyes from the SNAP product range (e.g., SNAP-Cell® 647-SiR and SNAP-Cell® TMR-Star) and detect them even at low levels during the recovery phase of the quench-pulse-chase protocol. Increased sensitivity also helped in the nanoscopy-based inspection of cellular lysate in Protocol 3 and allowed the visualization of granular structures made up of the SNAP-tagged viral protein. The combination of SNAP-based labeling and innovative detection via Airyscan has the potential to further bioimaging with higher resolution, sensitivity, and user-friendliness.


Figure 1. Overview of experimental protocols. This flowchart outlines the essential steps in Protocols 1, 2, and 3. For the definition of ‘stable CHIKV cells’, see Protocol 1, Procedure section.

Protocol 1: Pulse-chase experiments for long-term imaging with Nikon Ti2-E

Materials and Reagents

  1. 10-cm Petri dish (Corning, catalog number: 430167)
  2. Micropipette tips, serological pipettes, pipette aids, and microtubes for liquid handling
    1. TipOne® 1,000 µl XL (catalog number: S1122-1830)
    2. TipOne® 200 µl (catalog number: S1120-8810)
    3. TipOne® 20 µl filter tips (catalog number: S1120-1810) or SARSTEDT pipette tip 10 µl (catalog number: 70.1130.600)
    4. FisherbrandTM 5 ml serological pipets (catalog number: 13-676-10H)
    5. FisherbrandTM 10 ml serological pipets (catalog number: 13-676-10J)
    6. FisherbrandTM 25 ml serological pipets (catalog number: 13-676-10K)
    7. Drummond Pipet-Aid XL (catalog number: 4-000-205)
    8. Microtube 1.5 ml (SARSTEDT, catalog number: 72.690.001)
  3. Nunc 35-mm glass bottom (#1.5 or 0.16-0.19 mm thickness) dishes with 27 mm viewing area (Thermo Fisher Scientific, catalog number: 150682)
    Notes:
    1. Only open packages inside a biosafety cabinet and reseal any remaining dishes to avoid contamination.
    2. Use any dish or slide format that is compatible with an inverted microscope. Match the thickness of the glass bottom with the suggested thickness found on the microscope’s objective (typically #1.5 or 0.16-0.19 mm thickness). We prefer the dishes listed above because of their large viewing area. The cell lines used in this protocol can grow on glass substrates, but surface treatment (e.g., Poly-Lysine coating) may be necessary for other cell lines.
  4. HuH-7 cell line (Japanese Collection of Research Bioresources [JCBR], catalog number: JCBR 0403)
    Note: HuH-7 is a well-differentiated hepatocyte-derived cellular carcinoma cell line. It was originally taken from a liver tumor in a Japanese male in 1982 (Nakabayashi et al., 1982). The cells used in the creation of this protocol were obtained from John McLauchlan (Centre for Virus Research, Glasgow).
  5. A plasmid that encodes SNAPf®, a SNAP-tag protein (NEB, catalog number: N9183S; or as part of the SNAP-Cell® Starter Kit, NEB, catalog number: E9100S)
  6. For initial validation: plasmid that encodes pSNAPf-Cox8A control plasmid (also part of the SNAP-Cell® Starter Kit, NEB, catalog number: E9100S)
  7. Trypsin EDTA solution (Sigma, catalog number: T3924-500ML)
  8. Specific reagents for our cell line (for basic tissue culture), which we derived from the hepatoma cell line HuH-7
    1. Dulbecco's modified Eagle's medium (Sigma, catalog number: D6429-500ML)
    2. 100% Fetal Calf Serum (Gibco, catalog number: 10500-064)
    3. Gibco MEM Nonessential amino acids solution (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 11140050) 
    4. Gibco HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) buffer (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 15630130) 
  9. FluoroBrite DMEM, store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: A1896701)
  10. Cell-permeable SNAP-Cell® 647-SiR (New England Biolabs, catalog number: S9102S)
    Notes: 
    1. Package contains 30 nmol of the substrate. Resuspend with 50 µl of sterile dimethyl sulfoxide (DMSO, Fisher BioreagentsTM, catalog number: BP231-100) to make up a stock solution, which can be stored at -20 °C. We have used stock solutions that have been stored for up to 3 years. However, the manufacturer’s recommended shelf-life is three months dissolved in DMSO and two years dry.
    2. NEB also offers cell-impermeable SNAP probes (‘Cell Surface’ Probes); use these probes for studies of viral proteins that accumulate at the surface of host cells. 
  11. Optional: Red fluorescent or orange fluorescent cellular dye (e.g., MitoTrackerTM Orange CMTMRos, Thermo Fisher Scientific, catalog number: M7510)
    Note: To prepare a stock solution, dissolve lyophilized MitoTrackerTM probe in DMSO to a final concentration of 1 mM and store frozen and protected from light.
  12. Molecular Probes Invitrogen ProLong Live Antifade Reagent, for live cell imaging (Thermo Fisher Scientific, catalog number: P36975)
    Notes:
    1. Store at 2-8 °C for short-term storage and ≤ -20 °C for long-term storage.
    2. According to the manufacturer’s instructions, use the product within 30 days when stored at 2-8 °C. When stored at ≤ -20 °C, the product is stable for at least six months with up to four freeze-thaw cycles.
  13. Glutamax-I (Thermo Fisher Scientific, catalog number: 35050061)
  14. Live cell imaging solution (see Recipes)
  15. Tissue culture medium containing serum (see Recipes)

Equipment

  1. Air-displacement micropipettes
    1. Starlab ErgoOne® 100-1,000 µl Single-Channel Pipette (catalog number: S7110-1000) or Gilson F123602 PIPETMAN Classic Pipet P1000 (Fisher Scientific, catalog number: 10387322)
    2. Starlab ErgoOne® 20-200 µl Single-Channel Pipette (catalog number: S7100-2200) or Gilson F123615 PIPETMAN Classic Pipet P100 (Fisher Scientific, catalog number: 10442412)
    3. Starlab ErgoOne® 2-20 µl Single-Channel Pipette (catalog number: S7100-0220) or Starlab ErgoOne® 0.1-2.5 µl Single-Channel Pipette (catalog number: S7100-0125) or Gilson F144801 PIPETMAN Classic Pipet P2 (Fisher Scientific, catalog number: 10635313) or Gilson F123600 PIPETMAN Classic Pipet P20 (Fisher Scientific, catalog number: 10082012)
  2. Equipment for basic cell culture techniques and aseptic procedures, i.e.,
    1. Biosafety cabinet (e.g., Thermo Scientific Holten Safe 2010 Model 1.2, catalog number: 8207071100)
    2. Humidified incubator set to 37 °C (Panasonic Incusafe, catalog number: MCO-20AIC)
    Note: Any manufacturer and model of a biosafety cabinet will do, but the chosen biosafety cabinet needs to be appropriate for the containment of cells and viruses. When handling infectious viruses, use the proper facilities, practices, and procedures. Refer to local and international guidelines for laboratory biosafety. 
  3. -20 °C freezer (Labcold, catalog number: RLCF1520)
  4. Water bath (e.g., Clifton, catalog number: NE2-8D; however, any water bath set to 37 °C may be used)
  5. Table-top microcentrifuge, 4 °C to room temperature (RT), max speed ≥ 12,000 x g (Eppendorf, catalog number: 5424R)
    Note: For spinning down precipitate that may form during extended storage of SNAP probes.
  6. Live-cell imaging microscope for long-term imaging. For example, a Nikon Ti2-E inverted widefield microscope (for a similar setup, contact your local Nikon representative to create a customized order for the microscope system)
    1. Nikon Ti2-E inverted microscope stand
    2. Motorized stage (standard Ti2 encoded motorized XY stage)
    3. Lumencor Spectra X LED light source
    4. CFI Plan Apo Lambda 60x oil/1.4 NA objective
    5. Photometric Prime 95B sCMOS monochrome camera
    6. Semrock 32 mm filter (Green) for Nikon Ti2-E: GFP-4050B Filter cube with Ex 466/40 single-band bandpass filter (product code: FF01-466/40-25), DM495 dichroic beamsplitter (product code: FF495-Di03-25x36), BA525/50 single-band bandpass filter (product code: FF03-525/50-25)
    7. Semrock 32 mm filter (Red) for Nikon Ti2-E: Cy3-4040C Filter cube with Ex 531/40 single-band bandpass filter (product code: FF01-531/40-25), Dichroic Mirror DM 562 (product code: FF562-Di03-25x36, Barrier Filter: BA 593/40 (product code: FF01-593/40-25)
    8. Semrock 32 mm filter (Far-Red) for Nikon Ti2-E: Cy5-4040C Filter cube with Ex 628/40 single-band bandpass filter (product ID: FF02-628/40-25), Single Band Emitter (product ID: FF01-692/40-25), Single Band Dichroic (product ID: FF660-Di02-25x36) 
    9. Okolab stage top incubator, catalog number: H301-NIKON-NZ100/200/500-N. Set to 37 °C with 5% CO2 (any manufacturer and model that can be mounted on a Nikon Ti2 motorized stage will do)
    10. Perfect Focus System (PFS)
    Notes:
    1. The above list is not exhaustive but rather lists the essential components that have been useful in the creation of Protocol 1. Consult with your local Nikon representative for the remaining components of a customized microscope system.
    2. We prefer to image cells in open tissue-culture dishes with a coverslip-like glass bottom, which necessitates an inverted microscope stand. 
    3. As Protocol 1 is a powerful approach for long-term imaging of the labeled protein, it is important to keep cells in focus and correct for focus drift caused by thermal and mechanical conditions. This live-cell imaging system included Nikon’s fourth-generation Perfect Focus System (PFS), which monitors the partial reflection of a low-power infrared laser beam on the interface between the dish’s glass bottom and liquid media above. This feature provides continuous and real-time focus drift correction. 
    4. A large (25 mm x 25 mm) field of view (FOV) enables increased data throughput and capture of additional cells that would be outside the normal FOV. 
    5. We preferred an LED light source over laser-based illumination. LED illumination limited photobleaching and phototoxicity; ‘gentle’ imaging was essential during acquisition of five-dimensional datasets (5-D: multi-color 3-D Z-stacks over time). 
    6. A motorized microscope stage and the ability to record multiple positions during one experiment further increased the throughput of the imaging system.

Software

  1. Nikon NIS Elements AR imaging software
    Nikon NIS Elements AR imaging software controls all components of the microscope. Use NIS Elements to set-up parameters for acquisition of 5-D datasets. Users can also create rendered videos within NIS Elements and save individual frames of 5-D data. For our widefield images, we also used a Richardson-Lucy algorithm (set to 10 iterations) to deconvolve datasets within the Elements software.

Procedure

A published labeling procedure forms the basis of the protocol described here (Bodor et al., 2012). We build on this protocol by also describing a live-cell imaging setup that is suitable for long-term examination of protein turnover in five dimensions (i.e., 3-D multi-color fluorescence microscopy over time).

Note: Carry out all liquid handling steps that involve live cells inside the biosafety cabinet. Only use sterile pipettors, serological pipets, micropipettors, microtubes, and tips.

  1. Production of stable CHIKV cells
    We have previously used the HuH-7 hepatoma cell line to derive cells that stably harbor a modified CHIKV replicon; we designed this replicon to encode a fusion protein of CHIKV nsP3 and the SNAP-tag (Remenyi et al., 2018). We will refer to the cells from this secondary cell line as ‘stable CHIKV cells’ in the rest of the protocol. These cells also constitutively express the green fluorescent protein ZsGreen. 
    1. Use standard molecular cloning methods to generate N-, C-terminal, or internal SNAP-tag fusions. In our experimental system, we inserted the SNAP-tag within the C-terminal region of CHIKV nsP3 and added a flexible linker of Glycine amino acids at the N-terminal and C-terminal junctions (Remenyi et al., 2017). A plasmid that encodes SNAPf®, a SNAP-tag protein, is available from NEB (N9183S; or as part of the SNAP-Cell® Starter Kit, E9100S).
    2. After the SNAP-sequence has been inserted into a viral genome, evaluate whether modified viruses or replicons remain viable and whether the tagged protein can carry out the same biological function as the untagged protein. The methods for verification will vary depending on the virus and the protein-of-interest.
    3. Maintain cells in preferred tissue-culture format: we routinely passage our stable CHIKV cells in 10-cm Petri dishes. 
    4. We have developed this protocol with the stable CHIKV cell line, which we derived from HuH-7 cells. Other cell lines that support prolonged replication of non-cytotoxic CHIKV replicons include the C2C12 (mouse myoblast) cell line (Remenyi et al., 2018) and BHK-21 (baby hamster kidney) cell line (Utt et al., 2015). However, we have not yet validated Protocols 1-3 in these cell lines. 
  2. Detach stable CHIKV cells from the growth surface by adding enough trypsin to cover the cells that are attached to the surface of the respective culturing vessel (e.g., 1-2 ml of trypsin for 10-cm dishes) and seed into a 35-mm dish with coverslip bottom. Prepare a second and third dish containing positive and negative control.
    1. As a negative control, we seed naïve cells (known not to express SNAP-tag protein) in a second dish. 
    2. NEB’s SNAP-Cell Starter Kit also contains a positive control plasmid (pSNAPf-Cox8A Control Plasmid), which can be transfected into cells to produce SNAP-tagged cytochrome c oxidase with a well-characterized mitochondrial localization. Thus, the third dish may contain cells transfected with pSNAPf-Cox8A control plasmid or any plasmid encoding a SNAP-tagged protein with well-characterized subcellular localization. 
    Notes:
    1. For beginners, we recommend handling only three dishes at a time (one dish for the sample, one dish for the negative control, and one dish for the positive control) during the labeling phase of this protocol. Advanced users requiring higher throughput may consider 35-mm imaging dishes with four compartments (e.g., ibidi µ-Dish 35 mm Quad, catalog number 80416) instead of using individual dishes. Carry out simultaneous experiments (e.g., two sample conditions, one negative control and one positive control) in the subdivisions of the dish. 
    2. The exact amount of trypsin needed to dissociate adherent cells is dependent on the cell type and age of cells.
    3. We adjust the cell seeding density depending on the length of our desired chase period and the duplication time of the cell line we use for the experiment. 
    4. We typically aim for 60% to 80% confluency. For example, assuming a doubling time of 24 h and a desired chase period of 24 h, we would seed our SNAP-tagged cell line to 20% confluency, stain with SNAP-reagents the following day (= pulse, at 40% confluency) and image during a 24-h chase (allowing cells to reach 80% confluency during live-cell imaging).
  3. Incubate cells under standard growth conditions (i.e., 37 °C at 5% CO2) overnight. 
  4. On the next day, take the frozen stock solution of SNAP-Cell® 647 SiR from the -20 °C freezer and thaw at room temperature. 
  5. In a 1.5-ml microtube, dilute thawed SNAP-Cell® 647 SiR; perform a 1:1,000 (final concentration of 0.6 µM) dilution in complete cell culture media. Final volume should be at least 0.6 ml to cover the dish area above the coverslip. Users can increase the volume to 1 ml to reduce the risk of drying out the cells. Vortex briefly (for ≥ 5 s) or pipet the diluted labeling solution up and down (ten times). 
    1. SNAP-Cell® probes are cell-permeable. Our protocols label a fusion protein of CHIKV nsP3 and the SNAP-tag. This fusion protein localizes to intracellular compartments, and hence we only stain with cell-permeable probes.
    2. Other probes from NEB include SNAP-Cell® 505 (green fluorescent), SNAP-Cell® Oregon Green (green fluorescent), and SNAP-Cell® TMR-Star (red fluorescent). Optimize final concentrations for different SNAP-tagged proteins and cell lines; factors like protein abundance and non-specific binding may vary depending on the tagged protein or cell line. 
    3. According to the manufacturer, optimal substrate concentrations range from 1 to 20 µM, with best results usually obtained at concentrations between 1 and 5 µM. We have found that even 0.6 µM of SNAP-Cell® SiR provided sufficient staining. We do not prepare more media than we expect to consume within one hour. Always include a negative control (naïve cells known not to express SNAP-tag protein) when optimizing labeling conditions.
      Note: Recipe for three dishes (using 0.6 ml in Step 7, scale accordingly for more dishes): 1998 µl Complete Cell Culture Media; 2 µl SNAP-Cell® 647 SiR.
  6. Spin the diluted labeling solution for 5 min at maximum speed (≥ 12,000 x g) to remove possible insoluble fluorescent debris. Take care not to disturb the pellet when removing supernatant (which may be invisible).
  7. Replace the medium on stable CHIKV cells with 0.6-1 ml of SNAP-tag labeling medium (pre-heated to 37 °C in a water bath). Incubate for 15 min at 37 °C, 5% CO2.
    Note: At this step, the benzyl group on the SNAP-Cell® 647 SiR substrate will covalently link to the SNAP-tag and release guanine. We found that a 15-min incubation gave us optimal labeling. According to the manufacturer, optimal reaction times range from 5 to 30 min, respectively, depending on experimental conditions and expression levels of the SNAP-tagged protein. 
  8. During this 15-min incubation period, prepare a cold water bath containing ice and water. Remove a frozen aliquot of ProLong Live reagent from the -20 °C freezer and thaw the aliquot in the cold water bath. Do not exceed 37 °C while thawing or using the reagent. We keep the reagent in the ice bath until Step 10. 
  9. Wash the cells three times each with 2 ml of tissue culture medium containing serum (pre-heated to 37 °C). 
  10. Replace the regular cell-culture media with 2 ml of fresh live cell imaging solution, consisting of FluoroBrite DMEM, supplemented with fetal bovine serum (at a final concentration of 10%), HEPES, Glutamax-I, Nonessential Amino Acids, and ProLong Live Antifade Reagent (see Recipes).
  11. Place cells back into the incubator after the final wash. Incubate for another 30 min, 37 °C at 5% CO2.
    Notes:
    1. The primary purpose of this step is to reduce the background staining of the SNAP reagent. The background staining of some SNAP probes, such as SNAP Cell® TMR-Star can be problematic in some cell lines whereas less background staining is observed in others (Cole, 2014). If the background staining is an issue, we recommend reducing the labeling time, concentration of probe, or increasing the number of washes. Step 10 also starts the incubation with ProLong Live reagent. 
    2. The manufacturer’s instructions of the ProLong Live reagent suggest incubating cells in the dark for 15 min to 2 h. 
    3. In our experience, by the time the sample dish reaches the microscope, final imaging settings have been set up, and the actual image acquisition starts, the total incubation period of cells with media containing ProLong Live will be at least 1.5 h (~40 min for processing three dishes in Steps 10-12, ~20 min for transferring dishes from tissue culture facility to microscope, and ~30 min for setting up imaging conditions). 
  12. Wash cells as described in Step 9 with regular cell-culture media. Replace media with 2 ml of live cell imaging media (made up in Step 10).
    Note: We supplement the live cell imaging media with ProLong Live solution at this step if live-cell imaging does not exceed 24 h. The manufacturer does not recommend leaving ProLong Live solution on live cells for more than 24 h. We also calculate the chase period from the completion of this step, since it marks the last time point at which the fluorescent substrate can label SNAP-tagged proteins.
  13. Optional: Stain cells with red fluorescent or orange fluorescent cellular dye (e.g., MitoTrackerTM Orange CMTMRos). 
    1. Dilute 1 mM MitoTrackerTM stock solution to the final working concentration (25-500 nM) in ‘live cell imaging buffer’. 
    2. Remove media from dishes and add pre-warmed (37 °C) staining solution containing MitoTrackerTM probe.
    3. Return dishes to the humidified incubator and incubate for 15-45 min, at 37 °C with 5% CO2.
    4. After incubation period is complete, replace staining solution with fresh pre-warmed ‘live cell imaging buffer’. 
  14. Transfer the three dishes (sample, positive control, and negative control) to microscope area for live-cell imaging with Nikon Ti2-E system. The live-cell imaging setup for SNAP-tagged cells is similar to standard configurations for live-cell fluorescence imaging. 
    1. We recommend Nikon’s resource on ‘live cell imaging’ for an introduction on the appropriate microscope setup for timelapse imaging.
    2. For additional resources, contact your local Nikon representative for NIS Elements Training handouts on ‘Advanced Acquisition’ modes (i.e., Multi-channel, Multi-point, Timelapse, and Z-stack)
    3. For alternative live-cell imaging setups, refer to Bodor et al. (2012). 
    4. We used a widefield imaging setup for extended imaging of the same field-of-view. We obtained high-quality results with a Nikon Ti2-E system. 
    5. Several factors determined our preference for this system, namely (i) the Ti2-E is equipped with a unique perfect focus system (PFS) that automatically corrects focus drift in real time during a prolonged period of imaging (ii) imaging with an LED light source allows for gentler imaging compared to laser-based confocal systems (iii) multipoint Z-stacks can be acquired quickly as a result of faster device movement and image acquisition (iv) quick acquisition reduces overall light exposure and subsequent phototoxicity (v) the Ti2-E provides a large field of view (FOV), which captures a large amount of cells within one FOV, and (vi) multi-point acquisitions further increase the throughput of the system.
  15. Image cells with the preferred imaging system
    1. SNAP Cell® 647-SiR should have an excitation maximum at 645 nm and an emission maximum at 661 nm. 
    2. With the Nikon Ti2-E inverted microscope, we use standard filter settings for the Cy5 dye. Stable CHIKV cells also endogenously express the green fluorescent ZsGreen reporter protein, which has an excitation maximum of 493 nm and an emission peak at 505 nm (image with a standard GFP filter set). 
    3. The advantage of using the far-red SNAP Cell® 647-SiR is that additional labeling with a red fluorescent cellular dye (e.g., MitoTrackerTM Orange) and imaging with filter settings for Cy3 dye is possible. Figure 2 shows representative images from a timelapse series, in which we set the microscope to take Z-stacks every 15 min for a total of 24 h.


      Figure 2. Combination of 5-D imaging and pulse-chase experiments. A. We only show selected frames from a multi-position timelapse series, in which the microscope acquired Z-stacks every 15 min at eight positions. In this setup, each Z-stack (composed of 41 slices) was completed within 45 s, whereas it took 6.5 min to obtain eight positions. The images on the left display all channels with pseudo-colors (green: ZsGreen, yellow: MitoTrackerTM Orange, magenta: SNAP-nsP3). Note that granular structures labeled at 0 h were still present at 3.75 h. This continued presence indicated that these structures remained stable for hours. B. Selected frames from the same timelapse series, this time presented in volume view. The images on the left display all channels with pseudo-colors (green: ZsGreen, yellow: MitoTrackerTM Orange, magenta: SNAP-nsP3). 

Recipes

  1. Live cell imaging solution (enough to add 2 ml of solution to three 35-mm dishes in Steps 10, 12, and optional Step 13)
    2.7 ml Fetal Calf Serum
    270 µl HEPES
    270 µl Glutamax-I
    270 µl MEM Nonessential Amino Acids
    270-540 µl ProLong Live Antifade Reagent
    22.96-23.22 ml FluoroBrite DMEM
    The total volume of live cell imaging solution: 27 ml
  2. Tissue culture medium containing serum
    500 ml Dulbecco's modified Eagle's medium
    56.5 ml Fetal Calf Serum
    5.6 ml MEM Nonessential amino acids solution
    5.6 ml HEPES

Protocol 2: Quench-pulse-chase experiments paired with ZEISS LSM 880 Airyscan Microscopy

Materials and Reagents

  1. Micropipette tips, serological pipettes, pipette aids, and microtubes for liquid handling
    1. TipOne® 1,000 µl XL filter tips (catalog number: S1122-1830)
    2. TipOne® 200 µl filter tips (catalog number: S1120-8810)
    3. TipOne® 20 µl filter tips (catalog number: S1120-1810) or SARSTEDT pipette tip 10 µl (catalog number: 70.1130.600)
    4. FisherbrandTM 5 ml serological pipets (catalog number: 13-676-10H)
    5. FisherbrandTM 10 ml serological pipets (catalog number: 13-676-10J)
    6. FisherbrandTM 25 ml serological pipets (catalog number: 13-676-10K)
    7. Drummond Pipet-Aid XL (catalog number: 4-000-205)
    8. Microtube 1.5 ml (SARSTEDT, catalog number: 72.690.001)
    9. 15 ml Centrifuge Tubes, Conical, Sterile (Starlab, catalog number: E1415-0200)
  2. Glass slide (Academy, catalog number: N/A142)
  3. Filter paper (Whatman®, catalog number: 1001025)
  4. 24-well plates (CytoOne®, catalog number: CC7682-7524)
  5. Clean, sterile 13 mm coverslips (# 1.5) (Academy, catalog number: NPS16/1818)
  6. Parafilm® M (Bemis, catalog number: PM-996)
  7. Aluminum foil (Caterwrap, catalog number: AKL-300-030M)
  8. 10-cm dishes
  9. Stable CHIKV cells (see Protocol 1)
  10. Specific reagents for our cell line (for basic tissue culture), which we derived from the hepatoma cell line HuH-7:
    1. Dulbecco's modified Eagle's medium (Sigma, catalog number: D6429-500ML)
    2. 100% Fetal Calf Serum (Gibco, catalog number: 10500-064)
    3. Gibco MEM Non-essential amino acids solution (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 11140050)
    4. Gibco HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) buffer (100x), store at 4 °C up to 24 months from the date of manufacture (Thermo Fisher Scientific, catalog number: 15630130)
  11. Trypsin EDTA (Sigma, catalog number: T3924-500ML)
  12. PBS, Phosphate Buffered Saline (VWR Lifescience, catalog number: E404-100TABS)
  13. Antibody for immunolabeling of the SNAP-tagged protein
    Note: We use a rabbit antibody produced in-house that detects CHIKV nsP3 (Remenyi et al., 2017 and 2018). 
  14. Goat anti-rabbit IgG (H+L) Cross-Absorbed Secondary Antibody, DyLight® 405 (Thermo Fisher Scientific, Invitrogen, catalog number: 35551)
    Note: The combination of Dylight® 405, ZsGreen, and TMR-Star provides effective color separation and sensitivity for three-color imaging.
  15. SNAP-Cell® TMR-Star (New England Biolabs, catalog number: S9105S; or as part of the SNAP-Cell® Starter Kit, catalog number: E9100S)
    Note: Prepare stock solution as described for SNAP-Cell® 647-SiR.
  16. SNAP-Cell® Block (bromothenylpteridine, BTP, New England Biolabs, catalog number: S9106S; or as part of the SNAP-Cell® Starter Kit, catalog number: E9100S). Storage: -20 °C for at least three years dry or three months as a stock solution dissolved in DMSO
  17. Fixative, 4% Formaldehyde in phosphate buffered saline (PBS), pH 6.9
    Note: For the detailed protocol how to prepare the fixative, see: https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs. Briefly, prepare the fixative from Paraformaldehyde powder (Fisher Chemical, catalog number: T353-500). Dissolve paraformaldehyde powder in 1x sterile PBS (made from tablets) (VWR Lifescience, catalog number: E404-100TABS). Adjust the pH to 6.9 with diluted Hydrochloric Acid (HCl) (Fisher Chemical, catalog number: H/1100/PB17). For long-term storage, keep 15-ml aliquots at -20 °C. When needed, thaw aliquots (vortex thoroughly to remove any precipitation) and use for each experiment. Store any remaining solution for up to 1 month at 4 °C.
  18. Mounting Medium (ProLong Diamond Antifade Mountant, catalog number: P36965)
    Note: We use ProLong Diamond because it protects both fluorescent dyes (in our setup: SiR + DyLight® 405) and fluorescent proteins (in our setup: ZsGreen) from fading.
  19. Complete Cell Culture Media (see Recipes)

Equipment

  1. Micropipettes
    1. Starlab ErgoOne® 100-1,000 µl Single-Channel Pipette (catalog number: S7110-1000) or Gilson F123602 PIPETMAN Classic Pipet P1000 (Fisher Scientific, catalog number: 10387322)
    2. Starlab ErgoOne® 20-200 µl Single-Channel Pipette (catalog number: S7100-2200) or Gilson F123615 PIPETMAN Classic Pipet P100 (Fisher Scientific, catalog number: 10442412)
    3. Starlab ErgoOne® 2-20 µl Single-Channel Pipette (catalog number: S7100-0220) or Starlab ErgoOne® 0.1-2.5 µl Single-Channel Pipette (catalog number: S7100-0125) or Gilson F144801 PIPETMAN Classic Pipet P2 (Fisher Scientific, catalog number: 10635313) or Gilson F123600 PIPETMAN Classic Pipet P20 (Fisher Scientific, catalog number: 10082012)
  2. Metal tweezers with fine tips for lifting and handling glass coverslips (EMS, catalog number: 78316-1)
  3. Equipment for basic cell culture techniques and aseptic procedures, i.e.,
    1. Biosafety cabinet (e.g., Thermo Scientific Holten Safe 2010 Model 1.2, catalog number: 8207071100)
    2. Humidified incubator set to 37 °C (Panasonic Incusafe, catalog number: MCO-20AIC)
    Note: Any manufacturer and model of a biosafety cabinet will do, but the chosen biosafety cabinet needs to be appropriate for the containment of cells and viruses. When handling infectious viruses, use the proper facilities, practices, and procedures. Refer to local and international guidelines for laboratory biosafety.
  4. -20 °C freezer (Labcold, catalog number: RLCF1520)
  5. Water bath (e.g., Clifton, catalog number: NE2-8D; however, any water bath set to 37 °C may be used)
  6. Table-top microcentrifuge, 4 °C to room temperature (RT), max speed ≥ 12,000 x g (Eppendorf, model: 5424R)
    Note: For spinning down precipitate that may form during extended storage of SNAP probes.
  7. Safety glasses (any manufacturer or model that protects users from formaldehyde splashes will do)
  8. Confocal laser scanning microscope. For example, a customized ZEISS LSM 880 system that includes the following essential components (for a similar setup, contact your local ZEISS representative for exact ordering information as product codes may differ from customer to customer and country to country):
    1. Axio Imager Z2 stand, motorized (upright system), product ID: 430000-9902-000
    2. Motorized Stage, Scanning Stage 130x85 STEP, product ID: 432033-9902-00
    3. Mounting Frame 160x116 f/ Slides 76x26, product ID: 432315-0000-000
    4. Scan module LSM 880, product ID: 000000-1994-956
    5. Support f/scan module LSM (Imager Tube), product ID: 000000-1265-660
    6. Stepper motor control f, 2 Axes SMC2009, product ID: 432929-9011-000
    7. Real-time controller standard, product ID: 000000-2031-918
    8. Objective C PApo 63x/1.4 Oil DIC UV-IR, product ID: 421782-9900-799
    9. 488 Vis Laser, Laser Argon Multiline 25 mW, product ID: 000000-2086-081
    10. 561 Vis Laser, Laser 561 nm for LSM 710, product ID: 00000-1410-117
    11. 633 Vis Laser, Laser Rack LSM 880 incl. 633 Laser, product ID: 000000-2085-478
    12. Airyscan SR module GaAsP for LSM, product ID: 000000-2058-580
    13. Emission filter BP495-550 + LP570 for Airyscan, product ID: 000000-2070-488 
    14. Emission filter BP570-620 + LP645 for Airyscan, product ID: 000000-2070-489
    15. Double BP 420-480 + BP495-620 for Airyscan, product ID: 000000-2095-049
    16. Double BP 465-505 + LP525 for Airyscan, product ID: 000000-2095-051
    17. Double BP 420-480 + LP605 for Airyscan, product ID: 000000-2095-052 
    18. User PC advanced Z55A highend, product ID: 000000-2142-968
    19. ‘Airyscan Fast’ illumination module upgrade for 1x LSM 880 system
    Notes:
    1. The above list is not exhaustive but only lists essential components that we have found useful for our protocols. Consult with ZEISS about additional components to complete the microscope system (e.g., joystick for stage, hardware license keys, beam splitter, switching mirror, and nosepiece). 
    2. Follow the manufacturer’s recommendations for applying all necessary Airyscan settings within the ZEN microscope software. Our LSM 880 Airyscan microscope could provide a maximum lateral resolution of 140 nm and axial resolution of 400 nm for a fluorophore emitting at 480 nm (images processed with ZEN Black software). The resolution can increase even further to 120 nm XY and 350 nm Z resolution with the use of ZEN Blue software. 
    3. Our original system (LSM 880 with Airyscan) was retroactively upgraded to an ‘Airyscan Fast’ system, whereas today’s systems can be customized with an ‘Airyscan Fast’ module from the start. The ‘Airyscan Fast’ illumination system allows simultaneous illumination of 4 pixels simultaneously to allow very fast and gentle imaging of samples, including the option to scan at super-resolution with 1.5x resolution improvement in XY & Z, imaging at up to 19 images/second with 512 x 512 pixels, 27 images per second at 480 x 480, and 6 images per second at 1024 x 1024.

Software

  1. ZEISS ZEN software (ZEN 2.3 system HWL for FAST Airyscan)
    Note: ZEISS ZEN software drives all components of the LSM 880 system, including setup of Airyscan imaging. ZEN software can also acquire and process raw super-resolution image datasets; save processed image files as ‘.czi’ files, which can be exported to bioimaging analysis software (e.g., Icy software). Saving in this file format ensures the preservation of all the metadata associated with each imaging experiment. 
  2. Icy bioimaging software
    Note: Bioimaging analysis software. We recommend the free software Icy to visualize, annotate and quantify bioimaging datasets, which can be imported from ZEISS ZEN software packages (de Chaumont et al., 2012). We found that Icy has an intuitive user interface.

Procedure

We use a published labeling protocol (Bodor et al., 2012) to analyze ‘new’ (i.e., freshly translated) pools of the SNAP-tagged viral protein (SNAP-nsP3). By combining the labeling approach with a sensitive detection method, ZEISS LSM 880 Airyscan microscopy, we can visualize the re-emergence of ‘new’ protein and the intracellular sites where ‘new’ proteins accumulate. In the first step (called ‘quench’), a nonfluorescent SNAP-substrate covalently binds to the pool of SNAP-nsP3 present at the onset of an experiment (Figure 3, diagram). After a given amount of time (chase), a second, fluorescent substrate (pulse) labels the cells as described in Protocol 1 (Figure 3, diagram). The pulse only stains the protein pool synthesized during the chase period. Thus, ZEISS LSM 880 Airyscan microscopy will only make this ‘new’ (i.e., freshly translated) pool visible. The total pool of SNAP-nsP3 (pulsed pool + quench pool) can be stained with a standard immunofluorescence assay approach, which reveals the quenched pool that would otherwise remain undetected (Figure 3).

Note: Carry out all liquid handling steps that involve live cells or formaldehyde-containing wells inside the biosafety cabinet. Only use sterile pipettors, pipets, micropipettors, microtubes, and tips when working with live cells. Wear safety glasses when handling formaldehyde solutions.

  1. Place sterile coverslips in separate wells of a 24-well plate.
    Notes:
    1. Adjust the total number of wells according to the number of ‘post-chase’ timepoints. Also include a positive control (e.g., a well without any SNAP Cell® Block added) and negative control (e.g., a well without any chase period). 
    2. In this protocol: two post-chase timepoints (3 h, 6 h) and two controls (four wells in total).
  2. Detach stable CHIKV cells from growth surface by adding enough trypsin solution (pre-heated to 37 °C in a water bath) to cover the cells that are attached to the surface of the respective culturing vessel (e.g., 1-2 ml of trypsin solution for 10-cm dishes) and seed stable CHIKV cells in wells from Step 1.
    Seeding density will again depend on the total duration of the experiment and the doubling time of cells. We aim for about ~80% confluency at the time of fixation of the 0-h, 3-h, and 6-h samples. Thus, we also seed fewer cells in the 3-h and 6-h wells to account for the longer incubation times compared to the 0-h well (10-15% less in 3-h well and 20-30% less in 6-h well).
  3. Incubate at 37 °C, 5% CO2 overnight.
  4. On the next day, take the frozen stock solution of SNAP Cell® Block from the -20 °C freezer and thaw at room temperature. In a 1.5-ml microtube, dilute SNAP Cell® Block to a final concentration of 2 µM in complete media. Vortex briefly (for ≥ 5 s). Prepare > 200 µl per coverslip. Use diluted reagent within the hour.
  5. Replace media on cells with 200 µl of the SNAP Cell® Block diluted solution (pre-heated to 37 °C in a water bath) per well (three wells in total). Do not change media on the fourth well (this will serve as the ‘positive control’). Incubate for 30 min. 
  6. Wash cells three times with 1 ml of complete media (pre-heated to 37 °C in a water bath; this washes away any free substrate, which would interfere with downstream applications). Remove media after the last wash step.
  7. Add 1 ml of complete media (pre-heated to 37 °C in a water bath) to each well and place cells into a tissue-culture incubator for 30 min.
  8. Wash cells as in Step 6. To prevent the cells from drying out, do not remove the wash media after the last wash step. 
  9. Prepare a new 24-well plate and add 1 ml of 4% formaldehyde fixative to two of the wells. 
  10. Use forceps to transfer one of the ‘quenched’ coverslips and the ‘unblocked’ coverslip to the new plate and submerge in formaldehyde (make sure the side with the layer of cells remains up). The fixed cells on the ‘quenched’ coverslip will serve as the ‘no chase’ control. The cells on the ‘unblocked’ coverslip will serve as a positive control for SNAP-reagent staining.
  11. Place the original plate, which contains the remaining coverslips, back into the tissue-culture incubator. Incubate cells for the desired chase period of 3 h. 
  12. Place the new plate containing fixed coverslips at 4 °C for storage until all coverslips for all chase periods have been collected. Seal edges of the plate during storage to avoid excessive evaporation (strips of Parafilm M work well).

Note: If the quench was indeed complete, no labeling should occur during the ‘pulse’ period. If Airyscan microscopy can still detect unquenched SNAP-tagged protein, this indicates that the available SNAP Cell® Block reagent did not fully quench the pre-existing pool. Repeat experiments with an increased concentration of SNAP Cell® Block or with a prolonged incubation time until experimental conditions lead to complete quenching of SNAP-tagged protein.

  1. After three hours, transfer one coverslip to formaldehyde-containing well as described in Step 10 (remove the plate from 4 °C storage and add 1 ml of 4% formaldehyde to a third well). The fixed cells on this coverslip will serve as the ‘3-h chase’ sample. 
  2. Place the original plate, which contains the remaining coverslip, back into the tissue culture incubator. Incubate cells for another three hours. Place plate that holds fixed ‘no chase’ and ‘3-h chase’ coverslips back to 4 °C for storage.
  3. Repeat Steps 13 and 14 (transfer coverslips into a fourth, formaldehyde-containing well). The fixed cells on this last coverslip will serve as the ‘6-h chase’ sample. Incubate for 30 min at room temperature or for 1-2 h at 4 °C to complete the fixation process of ‘6-h chase’ sample.
  4. In the biosafety cabinet, wash ‘No chase’, ‘No quench’, ‘3-h chase’, and ‘6-h chase’ controls three times with PBS to remove the formaldehyde fixative.
    Note: At this point, users also have the option to handle 24-well plates outside a biosafety cabinet as the cells have already undergone chemical fixation. However, we still prefer to carry out Steps 17, 19, and 20 inside a biosafety cabinet to avoid the contamination of stock solutions and cell culture media.
  5. Take the frozen stock solution of SNAP Cell® TMR-Star from -20 °C freezer and thaw at room temperature. In a 1.5-ml microtube, dilute SNAP-Cell® TMR-Star; perform a 1:600 (final concentration of 1 µM) dilution in complete cell culture media. Prepare at least 200 µl per well to cover the entire coverslip. Vortex briefly (for ≥ 5 s) or pipet the diluted labeling solution up and down (ten times).
    Notes: 
    1. This protocol should also be compatible with SNAP-Cell® 647 SiR. Use the same labeling conditions as described in Protocol 1 (i.e., 1:1,000 dilution, 15 min incubation).
    2. Recipe for four coverslips in 24-well plate, scale according to the number of wells:
      998.4 µl Complete Cell Culture Media
      1.6 µl SNAP-Cell® TMR-Star
  6. Spin diluted labeling solution for 5 min at maximum speed (≥ 12,000 x g) to remove possible insoluble fluorescent debris. Take care not to disturb the pellet when removing supernatant (which may be invisible).
  7. Replace the medium on stable CHIKV cells with 200 µl of SNAP-tag labeling medium. Incubate for 15 min at 37 °C, 5% CO2. Protect samples from excessive light exposure during Steps 20-23 (e.g., wrap 24-well plate in aluminum foil).
    Note: We found that a 15-min incubation gave us optimal labeling. According to the manufacturer, optimal reaction times range from 5 to 30 min, respectively, depending on experimental conditions and expression levels of the SNAP-tagged protein.
  8. Wash the cells three times each with 1 ml of tissue culture medium containing serum (pre-heated to 37 °C).
  9. Use a preferred immunofluorescence assay protocol to stain the pool of total tagged protein. At this point, users can handle 24-well plates outside the biosafety cabinet–critical reagents in our case: primary antibodies from rabbit antiserum against nsP3 and dye-conjugated secondary antibodies (anti-rabbit DyLight® 405). 
  10. Mount the immunolabeled coverslips in preferred mounting medium (e.g., ProLong Diamond Antifade Mountant):
    1. Place a droplet of mountant on a glass slide, remove immunostained coverslips from 24-well plate using forceps, and slowly lower coverslip (cell side down) onto the droplet. 
    2. Use tissue or filter paper to wipe off any excess mountant. We usually fit two to three coverslips on a standard glass slide.
  11. Allow mountant to cure at room temperature and in the dark (overnight or longer). We store cured slides at 4 °C. 
  12. Image with ZEISS LSM 880 Airyscan microscopy
    1. SNAP Cell® TMR-Star should have an excitation maximum at 554 nm and an emission maximum at 580 nm. Set up three-color imaging with a blue, green, and red channel. Refer to ZEN software manuals for set-up of ZEISS LSM 880 Airyscan microscopy.
    2. Upon completion of image acquisition and processing, we use Icy bioimaging software to open saved and processed Airyscan data, which we saved in the .czi file format. Figure 3 shows representative images from a three-color imaging experiment, including screenshots of the Icy histogram viewer for each channel.


    Figure 3. Airyscan microscopy after chase period reveals pools of unblocked viral protein. Representative images from a quench-pulse-chase experiment. Note that the co-distribution of the total nsP3 pool (stained with anti-nsP3 antibody) and the new pool of nsP3 (stained with TMR-Star) suggested a lack of spatial separation of old and new pools of SNAP-nsP3. We set up the microscope for three-color imaging in the blue, green, and red channels. We adjusted image contrast within the Icy platform by dragging the adjustable bounds of the histogram viewer (marked by arrows), which enhanced the contrast in the selected channel without altering the data. We chose a viewing range that provided the best contrast for SNAP-nsP3 channel at the 3-h and 6-h timepoints. We also used the same viewing range to display the data from the positive and negative controls (‘Histogram range’ window, marked by asterisk *, view range minimum of 0 and maximum of 8469.0 pixel intensity values). Note that by applying the colormap ‘Fire’ (within Icy software) we could better display low-intensity granular structures. 

Recipes

  1. Complete Cell Culture Media
    500 ml Dulbecco's modified Eagle's medium
    56.5 ml Fetal Calf Serum
    5.6 ml MEM Nonessential amino acids solution
    5.6 ml HEPES

Protocol 3: Tracking SNAP-tagged viral protein assemblies in cell lysates with ZEISS LSM 880 Airyscan microscopy

Materials and Reagents

  1. Micropipette tips, serological pipettes, pipette aids, and microtubes for liquid handling:
    1. TipOne® 1,000 µl XL filter tips (catalog number: S1122-1830)
    2. TipOne® 200 µl filter tips (catalog number: S1120-8810)
    3. TipOne® 20 µl filter tips (catalog number: S1120-1810) or SARSTEDT pipette tip 10 µl (catalog number: 70.1130.600)
    4. FisherbrandTM 5 ml serological pipets (catalog number: 13-676-10H)
    5. FisherbrandTM 10 ml serological pipets (catalog number: 13-676-10J)
    6. FisherbrandTM 25 ml serological pipets (catalog number: 13-676-10K)
    7. Drummond Pipet-Aid XL (catalog number: 4-000-205)
    8. 1.5-ml Microtube (SARSTEDT, catalog number: 72.690.001)
    9. 15 ml Centrifuge Tubes, Conical, Sterile (Starlab, catalog number: E1415-0200)
  2. Parafilm® M (Bemis, catalog number: PM996)
  3. Aluminum foil (Caterwrap, catalog number: AKL-300-030M)
  4. Moistened paper (e.g., cut sheets of blot absorbent filter paper, Biorad, catalog number: 1703965)
  5. 10-cm Petri dish (Corning, catalog number: 430167)
  6. Ibidi 2-well µ-slide, an all-in-one chamber slide with polymer coverslip and ibiTreat surface for optimal cell adhesion (Ibidi, catalog number: 80286)
    Note: The IbiTreat surface modification makes the polymer coverslip surface hydrophilic. Alternative approaches to increase the adhesiveness of coverslip surfaces (i.e., Poly-Lysine treatment) and coated glass coverslips may be used (Wheeler et al., 2017). 
  7. Greiner CELLSTAR multiwall culture plates, 6-well (Merck/Sigma Aldrich, catalog number: Greiner 657160)
  8. Stable CHIKV cells (see Protocol 1)
  9. Trypsin EDTA (Sigma, catalog number: T3924-500ML)
  10. Materials and reagents for basic tissue culture (see Protocol 1 and 2)
  11. SNAP-Cell® TMR-Star (New England Biolabs, catalog number: S9105S; or as part of the SNAP-Cell® Starter Kit, catalog number: E9100S), Prepare stock solution as described for SNAP-Cell® 647-SiR
  12. PBS, Phosphate Buffered Saline (VWR Lifescience, catalog number: E404-100TABS)
  13. 4% Formaldehyde (see Protocol 2 Step 15)
  14. Triton X-100 (Sigma, catalog number: T924-500ml)
  15. KCl (Fisher Chemical, catalog number: P/4240/53)
  16. NaCl (Fisher Chemical, catalog number: S/3160/60)
  17. Magnesium chloride (MgCl2) hexahydrate 99.0-101.0%, VWR Chemicals, catalog number: 25108.260)
  18. Glycerol (Fisher Chemical, catalog number: G/0650/17)
  19. Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (Sigma, catalog number: P1851-100G)
  20. Leupeptin, hemisulfate salt ≥ 85% by HPLC (Sigma, catalog number: L8511-5MG)
  21. Pepstatin A (Sigma, catalog number: P5318-5MG)
  22. Aprotinin, from bovine lung (Sigma, catalog number: A6279-5ML)
  23. AEBSF (Pefabloc, Sigma, catalog number: 76307-100MG)
  24. NaOH (Fisher Chemical, catalog number: S/4920/60)
  25. Home-made Glasgow Lysis Buffer (GLB) containing protease inhibitors (see Recipes)
    1. 1x GLB, combine the stock solutions, Glycerol, Triton-X, and protease inhibitors (see Recipes)
    2. This protocol uses our laboratory’s preferred lysis buffer. We have not tried other lysis buffers that use NP-40 or SDS as detergents. However, note that 0.5% NP-40 is a component of the stress granule lysis buffer used to prepare cell lysates for stress granule core isolation (Wheeler et al., 2017). 
  26. Stock solutions for GLB (see Recipes)
  27. Stock solutions of protease inhibitors (see Recipes)
  28. 1x GLB (see Recipes)
  29. Tissue culture medium containing serum (see Recipes)

Note: For #14-24, alternatives of equal purity are also suitable.

Equipment

  1. Micropipettes
    1. Starlab ErgoOne® 100-1,000 µl Single-Channel Pipette (catalog number: S7110-1000) or Gilson F123602 PIPETMAN Classic Pipet P1000 (Fisher Scientific, catalog number: 10387322)
    2. Starlab ErgoOne® 20-200 µl Single-Channel Pipette (catalog number: S7100-2200) or Gilson F123615 PIPETMAN Classic Pipet P100 (Fisher Scientific, catalog number: 10442412)
    3. Starlab ErgoOne® 2-20 µl Single-Channel Pipette (catalog number: S7100-0220) or Starlab ErgoOne® 0.1-2.5 µl Single-Channel Pipette (catalog number: S7100-0125) or Gilson F144801 PIPETMAN Classic Pipet P2 (Fisher Scientific, catalog number: 10635313) or Gilson F123600 PIPETMAN Classic Pipet P20 (Fisher Scientific, catalog number: 10082012)
  2. Cell scrapers (Fisher Scientific, catalog number: 08-100-241)
  3. Equipment for basic cell culture techniques and aseptic procedures, i.e.,
    1. Biosafety cabinet (e.g., Thermo Scientific Holten Safe 2010 Model 1.2, catalog number: 8207071100) 
    2. Humidified incubator set to 37 °C (Panasonic Incusafe, catalog number: MCO-20AIC)
    Note: Any manufacturer and model of a biosafety cabinet will do, but the chosen biosafety cabinet needs to be appropriate for the containment of cells and viruses. When handling infectious viruses, use proper facilities, practices, and procedures. Refer to local and international guidelines for laboratory biosafety.
  4. -20 °C freezer (Labcold, catalog number: RLCF1520)
  5. Water bath (e.g., Clifton, catalog number: NE2-8D; however, any type of water bath set to 37 °C is appropriate)
  6. Table-top microcentrifuge, 4 °C to room temperature (RT), max speed ≥ 12,000 x g (Eppendorf, 5424R)
    Note: For spinning down precipitate that may form during extended storage of SNAP probes.
  7. Fume hood (any manufacturer or model that will protect users from formaldehyde fumes will do)
  8. Confocal laser scanning microscope. For example, a customized ZEISS LSM 880 system that includes the following essential components (for a similar setup, contact your local ZEISS representative for exact ordering information as product codes may differ from customer to customer and country to country):
    1. Axio Imager Z2 stand, motorized (upright system), product ID: 430000-9902-000 
    2. Motorized Stage, Scanning Stage 130x85 STEP, product ID: 432033-9902-00
    3. Mounting Frame 160x116 f/ Slides 76x26, product ID: 432315-0000-000
    4. Scan module LSM 880, product ID: 000000-1994-956
    5. Support f/scan module LSM (Imager Tube), product ID: 000000-1265-660
    6. Stepper motor control f, 2 Axes SMC2009, product ID: 432929-9011-000
    7. Real-time controller standard, product ID: 000000-2031-918
    8. Objective C PApo 63x/1.4 Oil DIC UV-IR, product ID: 421782-9900-799
    9. 488 Vis Laser, Laser Argon Multiline 25 mW, product ID: 000000-2086-081
    10. 561 Vis Laser, Laser 561nm for LSM 710, product ID: 00000-1410-117
    11. 633 Vis Laser, Laser Rack LSM 880 incl. 633 Laser, product ID: 000000-2085-478
    12. Airyscan SR module GaAsP for LSM, product ID: 000000-2058-580
    13. Emission filter BP495-550 + LP570 for Airyscan, product ID: 000000-2070-488 
    14. Emission filter BP570-620 + LP645 for Airyscan, product ID: 000000-2070-489
    15. Double BP 420-480 + BP495-620 for Airyscan, product ID: 000000-2095-049
    16. Double BP 465-505 + LP525 for Airyscan, product ID: 000000-2095-051
    17. Double BP 420-480 + LP605 for Airyscan, product ID: 000000-2095-052 
    18. User PC advanced Z55A highend, product ID: 000000-2142-968
    19. Transmitted light detector T-PMT, product ID: 000000-2014-999
    20. ‘Airyscan Fast’ illumination module upgrade for 1x LSM 880 system 
    Notes:
    1. The above list is not exhaustive but only lists essential components that we have found useful for our protocols. Consult with ZEISS about additional components to complete the microscope system (e.g., joystick for stage, hardware license keys, beam splitter, switching mirror and nosepiece).
    2. Follow the manufacturer’s recommendations for applying all necessary Airyscan settings within the ZEN microscope software. Our Airyscan microscope could provide a maximum lateral resolution of 140 nm and axial resolution of 400 nm for a fluorophore emitting at 480 nm (images processed with ZEN Black software). Airyscan resolution can increase even further to 120 nm XY and 350 nm Z resolution with the use of ZEN Blue software. 
    3. Our original system (LSM 880 with Airyscan) was retroactively upgraded to an ‘Airyscan Fast’ system, whereas today’s systems can be customized with an ‘Airyscan Fast’ module from the start. The ‘Airyscan Fast’ illumination system allows simultaneous illumination of 4 pixels simultaneously to allow very fast and gentle imaging of samples, including the option to scan at super resolution with 1.5x resolution improvement in XY & Z, imaging at up to 19 images/second with 512 x 512 pixels, 27 images per second at 480 x 480, and 6 images per second at 1024 x 1024.

Software

  1. ZEISS ZEN software (ZEN 2.3 system HWL for FAST Airyscan)
    Note: ZEISS ZEN software drives all components of the LSM 880 system, including setup of Airyscan imaging. ZEN software can also acquire and process raw super-resolution image datasets; save processed image files as ‘.czi’ files, which can be exported to bioimaging analysis software (e.g., Icy software). Saving in this file format ensures the preservation of all the metadata associated with each imaging experiment. 
  2. Icy bioimaging software
    Note: Bioimaging analysis software. We recommend the free software Icy to visualize, annotate and quantify bioimaging datasets, which can be imported from ZEISS ZEN software packages (de Chaumont et al., 2012). We found that Icy has an intuitive user interface.

Procedure

This protocol uses fluorescence light microscopy to reveal stable assemblies of SNAP-nsP3 protein that persist in cell lysates. It is partly based on an isolation protocol (Wheeler et al., 2017) that has been used to determine the proteome and substructure of stress granules (Jain et al., 2016). Our protocol adds additional flexibility in labeling (by using the SNAP labeling system) and detection (by using the sensitive ZEISS LSM 880 Airyscan confocal imaging system).

Note: Carry out all liquid handling steps that involve live cells inside the biosafety cabinet. Only use sterile pipettors, pipets, micropipettors, microtubes, and tips. After cell lysis, users can handle samples outside the biosafety cabinet. Wear safety glasses when handling formaldehyde solutions.

  1. Detach stable CHIKV cells with trypsin and seed in at least one well of a 6-well microtiter plate. Also, detach and seed naïve cells, which do not express SNAP-tagged proteins, in at least one well; these cells will serve as a negative control.
  2. Incubate cells under standard growth conditions (i.e., 37 °C at 5% CO2) overnight.
  3. On the next day, take frozen SNAP Cell® TMR-Star from -20 °C freezer and thaw at room temperature. In a 1.5-ml microtube, dilute SNAP-Cell® TMR-Star 1:600 (final concentration of 1 µM) dilution in complete cell culture media. Prepare at least 1 ml per well to cover the entire area of the well. Vortex briefly (for ≥ 5 s) or pipet the diluted labeling solution up and down (ten times). We have only validated this protocol with the SNAP-Cell® TMR-Star reagent. However, the protocol should also be compatible with SNAP-Cell® 647 SiR. Use the same labeling conditions as described in Protocol 1 (i.e., 1:1,000 dilution, 15 min incubation).
    Note: Recipe for two dishes, scale accordingly for additional dishes:
               1996.8 µl Complete Cell Culture Media
               3.2 µl SNAP-Cell® TMR-Star
  4. Spin diluted labeling solution for 5 min at maximum speed (≥ 12,000 x g) to remove possible insoluble fluorescent debris. Take care not to disturb the pellet when removing supernatant (which may be invisible).
  5. Replace the medium on stable CHIKV cells with 1 ml of SNAP-tag labeling medium (pre-heated to 37 °C in a water bath). Also, replace medium in the ‘negative control’ well. Incubate for 15 min at 37 °C, 5% CO2.
    Note: We found that a 15-min incubation gave us optimal labeling. According to the manufacturer, optimal reaction times range from 5 to 30 min, respectively, depending on experimental conditions and expression levels of the SNAP-tagged protein.
  6. Wash the cells three times each with 2 ml of tissue culture medium containing serum (pre-heated to 37 °C in a water bath). Do not remove media after the final wash.
  7. Place cells back into the humidified incubator for another 45-60 min.
    We use an extended wash-out period (compared to Protocol 1) to reduce the non-specific background-binding further. Moreover, we do not add ProLong Gold Live in this protocol, as live-cell imaging is only limited to an optional quality-control-step in Step 8.
  8. Optional: Confirm fluorescent staining of SNAP-nsP3 with microscopy
    1. A basic widefield fluorescent microscope equipped with a 10x or 20x objective is sufficient to evaluate the quality of SNAP-labeling through the microscope eyepiece. If documentation of staining quality is needed, take pictures with a connected camera. Also, confirm the absence of staining in the well that contains negative control. 
    2. We recommend this step when testing labeling conditions for the first time. Perform imaging quickly and proceed to Step 9 as soon as possible to limit the deterioration of cell health. If this is not logistically possible, we recommend proceeding to Step 9 directly.
  9. Replace complete cell media with 1 ml PBS and use separate cell scrapers to detach cells from growth area in each well. Transfer the resulting cell suspension to a 1.5 ml microtube. Pellet cells at 1,500 x g, 3 min at room temperature. Remove supernatant.
    Optional pause point: We freeze pellets at ≤ -20 °C if we want to carry out lysis at a later time.
  10. Lyse pellets by adding 300 µl of ice-cold Glasgow Lysis buffer containing protease inhibitors (see Recipes). Ensure complete re-suspension of pellet through repeated pipetting, flicking the tube, or vortexing. If pellet came from the freezer, thaw on ice for 5 min before adding lysis buffer. 
  11. Vortex Lysates for 30 s. Place on ice for 30 s. We return samples to ice in between vortexing cycles to prevent an extended incubation period at room temperature. Alternatively, lysates can be vortexed in a cold room.
  12. Repeat Step 11 three times.
  13. Spin at 850 x g for 5 min at 4 °C to remove remaining cellular debris.
    Note: We do not further purify assemblies of SNAP-nsP3 after this step but instead use the crude supernatant in the microscopic analysis. It would be interesting to test whether subsequent centrifugation at 18,000 x g can pellet assemblies of SNAP-nsP3; this step is essential in the isolation of a pure population of stress granule cores (Wheeler et al., 2017). After this step, users can handle cell lysates outside a biosafety cabinet.
  14. Transfer the entire volume of supernatant to a two-well Ibidi chambered plastic slide. Discard pelleted cellular debris.
    Note: We have noticed that the supernatant may appear turbid at this step. Proteins that are components of RNPs are known to undergo liquid-liquid phase transitions, and turbidity is due to the formation of small protein-rich droplets (Molliex et al., 2015). However, we have not yet tested whether purified SNAP-nsP3 can form similar droplets in solution.
  15. Incubate overnight at 4 °C in a humidified environment in the dark.
    1. To obtain humidified conditions, we cover the Ibidi slide with the supplied plastic cover and place the slide in a 10-cm Petri dish that also contains moistened paper (e.g., cut sheets of Western blotting filter). 
    2. We further seal dishes with Parafilm M and wrap them in aluminum foil to prevent light exposure. We incubate overnight to give assemblies of SNAP-nsP3 enough time to settle to the bottom of the chamber. 
    3. Future experiments may determine the minimum duration for deposition of SNAP-nsP3 to the bottom of the chambered slide; this information would be useful for reducing the overall time required to complete Protocol 3. 
  16. In a fume hood, add 1 ml of 4% formaldehyde to wells containing cell lysates. Incubate for one hour at room temperature.
    Note: Slowly dispense formaldehyde with a micropipette. We add this step to fix assemblies of SNAP-nsP3; fixation reduces the likelihood that the assemblies detach from the well surface during subsequent washing steps. 
  17. Wash wells three times with PBS. Ensure that wells do not dry out in between washes. 
  18. Transfer slide to microscopy system of choice:
    As described in Protocol 2, we use the ZEISS LSM 880 imaging system operated in Airyscan mode. Use the appropriate filter settings for TMR-Star, which should have an excitation maximum at 554 nm and an emission maximum at 580 nm. Also, acquire an image in brightfield mode. This image can provide an additional record of the examined biostructure.
  19. We recommend taking at least three images for each experiment:
    We acquire images with the 63x objective. Export processed Airyscan files to Icy bioimaging software. Adjust the image contrast within the Icy software by dragging the boundaries of the viewing range in the software’s histogram viewer (see Figure 4, arrows). We prefer viewing the SNAP-nsP3 channel with the ‘Fire’ colormap. Use Icy’s controls to zoom into regions-of-interest. See Figure 4 for representative images.


    Figure 4. Analysis of cell lysates with Airyscan microscopy. We processed samples according to Protocol 3 and detected granular structures containing TMR-Star-labelled SNAP-nsP3. We chose the ‘Fire’ colormap in Icy to display signals from the TMR-Star staining. We also adjusted image contrast by dragging the adjustable bounds of the histogram viewer (marked by arrows). We acquired images at a zoom factor of 1 with a 63x objective. During image processing, we also magnified regions-of-interest (ROI) by increasing the digital zoom within the Icy software (i.e., by a factor of 3.3 for ROI1 and 20 for ROI2). Lastly, we also acquired a brightfield channel to provide a reference and reveal all contrast-producing structures present in the lysate.

Recipes

  1. Home-made Glasgow Lysis Buffer (GLB) with protease inhibitors, 5 ml volume


  2. Stock solutions for GLB
    Add the appropriate amount of solids to distilled H2O to make stock solutions of PIPES (adjust pH to 7.2 with 3 M NaOH), KCl, NaCl, MgCl2 at concentration listed in the table. Store these stock solutions at 4 °C.
  3. Stock solutions of protease inhibitors, store in the following manner:
    Leupeptin 1 mg/ml at -20 °C
    Pepstatin A 1 mg/ml at -20 °C
    Aprotinin 2 mg/ml at 4 °C
    AEBSF (Pefabloc) 100 mM at 4 °C
  4. 1x GLB
    Combine the stock solutions, Glycerol, Triton-X, and protease inhibitors according to the table. Adjust volume to 5 ml with distilled H2O and pH to 7.2 with 3 M NaOH
    Note: Use fresh 1x GLB for each experiment.
  5. Tissue culture medium containing serum
    500 ml Dulbecco's modified Eagle's medium
    56.5 ml Fetal Calf Serum
    5.6 ml MEM Nonessential amino acids solution
    5.6 ml HEPES

Data analysis

We consider the unprocessed image files that microscope users save in either the Nikon NIS Elements AR software (.nd2 file format) or ZEISS ZEN software (.czi file format) to be ‘raw’ data. Thus, data analysis in these protocols was for qualitative purposes and made up exclusively of image visualization and digital processing of acquired images (e.g., adjusting brightness, contrast, and pseudo-colors) in either Nikon NIS Elements AR software or Icy bioimaging software. Although it is also possible to extract quantitative data from these images, our initial method development, which focused on the application of new labeling and imaging methods, did not include these types of bioimage-informatics approaches. Future studies may benefit greatly from incorporating rigorous bioimage-informatics techniques to the protocol described here.

Acknowledgments

A Wellcome Trust Investigator Award funded this work (WT 096670, awarded to Mark Harris). Purchase of shared equipment was made possible by a Wellcome Trust Multi-user equipment award (Zeiss LSM 880 instrument, WT104918MA, ‘Multifunctional imaging of living cells for biomedical sciences’). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. We thank Dr. Sally Boxall and the Bio-imaging Facility within the Faculty of Biological Sciences of the University of Leeds for access and help with Airyscan microscopes. The authors acknowledge Gina Gamble and Dr. Kate Lewis for their help using the Nikon Ti2-E Inverted microscope. We recognize the previous study by Bodor et al. (2012) which described the pulse-chase and quench-pulse-chase approaches. We are grateful to Mark B. Carascal and Dr. Samuel Ko for comments during the revision of the manuscript.

Competing interests

The authors declare that no conflicts of interest or competing interests exist.

References

  1. Bodor, D. L., Rodriguez, M. G., Moreno, N. and Jansen, L. E. (2012). Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging. Curr Protoc Cell Biol Chapter 8: Unit8.8. 
  2. Cole, N. B. (2014). Site-specific protein labeling with SNAP-tags. Curr Protoc Protein Sci 73: Unit 30.1. 
  3. de Chaumont, F., Dallongeville, S., Chenouard, N., Herve, N., Pop, S., Provoost, T., Meas-Yedid, V., Pankajakshan, P., Lecomte, T., Le Montagner, Y., Lagache, T., Dufour, A. and Olivo-Marin, J. C. (2012). Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9(7): 690-696. 
  4. Frigault, M. M., Lacoste, J., Swift, J. L. and Brown, C. M. (2009). Live-cell microscopy–tips and tools. J Cell Sci 122(Pt 6): 753-767. 
  5. Huff, J., Bergter, A., Birkenbeil, J., Kleppe, I., Engelmann, R. and Krzic, U. (2017). The new 2D Superresolution mode for ZEISS Airyscan. Nat Methods 14: 1223.
  6. Huff, J. (2015). The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution. Nat Methods 12: 1205.
  7. Jain, S., Wheeler, J. R., Walters, R. W., Agrawal, A., Barsic, A. and Parker, R. (2016). ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164(3): 487-498. 
  8. Kedersha, N., Stoecklin, G., Ayodele, M., Yacono, P., Lykke-Andersen, J., Fritzler, M. J., Scheuner, D., Kaufman, R. J., Golan, D. E. and Anderson, P. (2005). Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169(6): 871-884. 
  9. Kedersha, N., Tisdale, S., Hickman, T. and Anderson, P. (2008). Real-time and quantitative imaging of mammalian stress granules and processing bodies. Methods Enzymol 448: 521-552.
  10. Keppler, A., Kindermann, M., Gendreizig, S., Pick, H., Vogel, H. and Johnsson, K. (2004a). Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro. Methods 32(4): 437-444. 
  11. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. and Johnsson, K. (2004b). Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101(27): 9955-9959.
  12. Li, C., Kuang, C. and Liu, X. (2018). Prospects for fluorescence nanoscopy. ACS Nano 12(5): 4081-4085. 
  13. Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A. P., Kim, H. J., Mittag, T. and Taylor, J. P. (2015). Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163(1): 123-133. 
  14. Muller, C. B. and Enderlein, J. (2010). Image scanning microscopy. Phys Rev Lett 104(19): 198101. 
  15. Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T., Sato, J. (1982). Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 42(9): 3858-63.
  16. Remenyi, R., Gao, Y., Hughes, R. E., Curd, A., Zothner, C., Peckham, M., Merits, A. and Harris, M. (2018). Persistent replication of a chikungunya virus replicon in human cells is associated with presence of stable cytoplasmic granules containing nonstructural protein 3. J Virol 92(16). 
  17. Remenyi, R., Roberts, G. C., Zothner, C., Merits, A. and Harris, M. (2017). SNAP-tagged chikungunya virus replicons improve visualisation of non-structural protein 3 by fluorescence microscopy. Sci Rep 7(1): 5682. 
  18. Sheppard, C. J., Mehta, S. B. and Heintzmann, R. (2013). Superresolution by image scanning microscopy using pixel reassignment. Opt Lett 38(15): 2889-2892. 
  19. Utt, A., Das, P. K., Varjak, M., Lulla, V., Lulla, A., Merits, A. (2015). Mutations conferring a noncytotoxic phenotype on chikungunya virus replicons compromise enzymatic properties of nonstructural protein 2. J Virol 89(6): 3145-3162. 
  20. Wheeler, J. R., Jain, S., Khong, A. and Parker, R. (2017). Isolation of yeast and mammalian stress granule cores. Methods 126: 12-17.

简介

先进的标记技术使研究人员能够研究完整细胞内的蛋白质周转,并跟踪下游应用中的标记蛋白质。在病毒感染的背景下,病毒蛋白的成像和荧光标记的组合揭示了它们的生物活性和与宿主细胞的相互作用。最初的方法已将荧光蛋白如绿色荧光蛋白(GFP)融合到感兴趣的病毒蛋白中。相反,自标记酶标签,例如商业SNAP标签,人O 6 - 烷基鸟嘌呤-DNA-烷基转移酶的修饰形式,共价连接合成配体,用户可以根据需要添加。这里介绍的前两个协议建立在先前公布的脉冲追踪和猝灭脉冲追踪实验荧光标记协议之上。荧光标记与高级光学显微镜的组合可视化完整哺乳动物细胞中SNAP标记的病毒蛋白的动态转换。第三个方案还概述了如何在显微镜下检查细胞裂解物中标记的病毒蛋白的耐洗涤剂组装。这些方案展示了基于SNAP的标记系统的灵活性,用于追踪活细胞,完整固定细胞和细胞裂解物中感兴趣的病毒蛋白。此外,该方案采用最近开发的商业显微镜(例如,Airyscan显微镜),其平衡分辨率,速度,光毒性,光漂白和易用性。
【背景】为了更好地了解病毒蛋白在感染性生命周期中的作用,我们采用了现有的策略来促进细胞内蛋白质定位的确定,完整细胞中蛋白质动态的评估,以及裂解后的病毒蛋白的持续跟踪。宿主细胞。前两个方案建立在先前公布的按需SNAP标记和蛋白质周转分析方案上(Bodor et al。,2012)。在以前的工作中,我们的实验室已将基孔肯雅病毒(CHIKV)的病毒感染蛋白非结构蛋白3(nsP3)与SNAP标签融合,后者是20kDa单体DNA修复酶的修饰形式(Remenyi et al。,2017和2018)。在SNAP标记期间,添加合成的O 6 - 苄基鸟嘌呤(BG)衍生物导致SNAP标签中的反应性半胱氨酸残基与BG探针之间的共价键(Keppler 等人。,2004a和2004b)。

我们还将SNAP标记与用于通过光学显微镜检查细胞裂解物的方案相结合,这使得能够可视化耐洗涤剂的蛋白质组件。类似的方法使研究人员能够检测细胞裂解液中GFP标记的应力 - 颗粒蛋白的稳定颗粒组装(Jain et al。,2016; Wheeler et al。,2017) 。应激颗粒是RNA和蛋白质(RNP)的集合体,其在细胞应激条件下形成(Kedersha et al。,2005)。现在可以从酵母和哺乳动物细胞中分离出更稳定的应力颗粒核心(Jain et al。,2016)。 SNAP标记提供了一种跟踪标记的病毒蛋白的替代方法,这些蛋白可能存在于类似的亚细胞组装中。因此,方案3不仅可用于研究病毒蛋白的生物化学性质,还可用于跟踪存在于非膜状细胞器中的任何细胞蛋白,例如RNP和应激颗粒。例如,将SNAP标签整合到产生荧光标记的应力颗粒的细胞系的开发中(Kedersha et al。,2008)可以在这些细胞亚群的动态和定量成像期间增加实验灵活性。车厢。

我们的三种方案还利用了最近开发的用于多色荧光显微镜的商业成像系统。我们使用活细胞成像分析方案1中的标记样品(图1),因此建议遵循控制温度,降低光毒性,限制光漂白和维持细胞活力的一般程序(Frigault et al。,2009) )。选择的2018尼康Ti-E2系统使我们能够使用专有的完美聚焦系统(PFS)对大视场进行成像,减少聚焦漂移,并在电动载物台上记录多个位置。此外,与激光扫描共聚焦显微镜的典型成像设置相比,使用LED照射和不超过1秒的光照可以实现更温和的成像。

为了分析协议2和3中的标记蛋白质,我们选择了采用ZEISS Airyscan专有技术的共焦成像装置(图1),这是“图像扫描显微镜”方法的商业版本(Muller和Enderlein,2010; Sheppard et al。,2013)。 Airyscan显微镜代表了荧光超分辨率显微镜最近的一项创新,也被称为纳米镜(Li et al。,2018)。 Airyscan技术通过改进的探测器设计提高了系统分辨率,该探测器设计采用32通道探测器阵列(Huff,2015)。借助Airyscan的全新2D超分辨率模式,这种检测方法现在可以将分辨率提高2倍,同时降低所需的荧光强度,从而获得高质量的图像(Huff et al。,2017)。 Airyscan系统允许我们使用SNAP产品系列中的荧光染料对样品进行染色(例如,SNAP-Cell ® 647-SiR和SNAP-Cell ®< / sup> TMR-Star)并且在猝灭脉冲追踪协议的恢复阶段期间甚至在低水平检测它们。增加的灵敏度也有助于在方案3中基于纳米检查的细胞裂解物的检查,并允许可视化由SNAP标记的病毒蛋白组成的颗粒结构。基于SNAP的标记和通过Airyscan进行的创新检测相结合,可以进一步提高生物成像的分辨率,灵敏度和用户友好性。


图1.实验方案概述。此流程图概述了方案1,2和3中的基本步骤。有关“稳定CHIKV细胞”的定义,请参阅方案1,程序部分,

关键字:融合蛋白, 应激颗粒, RNPs, 特定位点的蛋白质标记, 化学标记, 细胞生物学, 生物成像, 活细胞成像, 基孔肯雅热病, CHIKV, 甲病毒, 虫媒病毒, nsP3

方案1:使用Nikon Ti2-E进行长期成像的脉冲追踪实验

材料和试剂

  1. 10厘米培养皿(Corning,目录号:430167)
  2. 微量移液器吸头,血清移液器,移液器辅助设备和用于液体处理的微管
    1. TipOne ®1,000μlXL(目录号:S1122-1830)
    2. TipOne ®200μl(目录号:S1120-8810)
    3. TipOne ®20μl过滤嘴(目录号:S1120-1810)或SARSTEDT移液器吸头10μl(目录号:70.1130.600)
    4. Fisherbrand TM 5 ml血清移液管(目录号:13-676-10H)
    5. Fisherbrand TM 10 ml血清移液管(目录号:13-676-10J)
    6. Fisherbrand TM 25 ml血清移液管(目录号:13-676-10K)
    7. Drummond Pipet-Aid XL(目录号:4-000-205)
    8. Microtube 1.5ml(SARSTEDT,目录号:72.690.001)
  3. Nunc 35毫米玻璃底(厚度为#1.5或0.16-0.19毫米),观察区域为27 mm(Thermo Fisher Scientific,目录号:150682)
    注意:
    1. 仅在生物安全柜内打开包装并重新密封剩余的餐具以避免污染。
    2. 使用与倒置显微镜兼容的任何碟形或幻灯片格式。使玻璃底部的厚度与显微镜物镜上的建议厚度相匹配(通常为#1.5或0.16-0.19 mm厚度)。我们更喜欢上面列出的菜肴,因为它们的观赏面积很大。该方案中使用的细胞系可以在玻璃基质上生长,但其他细胞系可能需要进行表面处理( ,聚赖氨酸涂层)。
  4. HuH-7细胞系(日本研究生物资源集合[JCBR] ,目录号:JCBR 0403)
    注意:HuH-7是一种分化良好的肝细胞来源的细胞癌细胞系。它最初取自1982年日本男性的肝肿瘤(Nakabayashi et al。 ,1982)。用于创建该方案的细胞来自John McLauchlan(格拉斯哥病毒研究中心)。
  5. 编码SNAPf ®的质粒,SNAP标签蛋白(NEB,目录号:N9183S;或作为SNAP-Cell ® Starter Kit的一部分,NEB,目录号:E9100S)
  6. 初步验证:编码pSNAP f -Cox8A对照质粒的质粒(也是SNAP-Cell ® Starter Kit,NEB,目录号:E9100S的一部分)
  7. 胰蛋白酶EDTA溶液(Sigma,目录号:T3924-500ML)
  8. 我们的细胞系(用于基础组织培养)的特异性试剂,我们从肝细胞瘤细胞系HuH-7中获得
    1. Dulbecco改良的Eagle's培养基(Sigma,目录号:D6429-500ML)
    2. 100%胎牛血清(Gibco,目录号:10500-064)
    3. Gibco MEM非必需氨基酸溶液(100x),自生产之日起至4个月内储存至24个月(Thermo Fisher Scientific,目录号:11140050)&nbsp;
    4. Gibco HEPES(N-2-羟乙基哌嗪-N-2-乙磺酸)缓冲液(100x),自制造之日起至4个月保存至24个月(Thermo Fisher Scientific,目录号:15630130)&nbsp;
  9. FluoroBrite DMEM,自生产之日起24个月内储存于4°C(Thermo Fisher Scientific,目录号:A1896701)
  10. 细胞渗透性SNAP-Cell ® 647-SiR(New England Biolabs,目录号:S9102S)
    注意:&nbsp;
    1. 包含30nmol的底物。用50μl无菌二甲基亚砜(DMSO,Fisher BioreagentsTM,目录号:BP231-100)重悬以制备储备溶液,其可以在-20℃下储存。我们使用的库存解决方案已存储长达3年。但是,制造商推荐的保质期是在DMSO中溶解三个月,干燥两年。
    2. NEB还提供细胞不可渗透的SNAP探针('细胞表面'探针);使用这些探针研究在宿主细胞表面积聚的病毒蛋白质。
  11. 可选:红色荧光或橙色荧光细胞染料(例如,MitoTracker TM Orange CMTMRos,Thermo Fisher Scientific,目录号:M7510)。
    注意:要制备储备溶液,将冻干的MitoTracker TM 探针溶解在DMSO中至终浓度为1 mM并储存冷冻避光。
  12. Molecular Probes Invitrogen ProLong活抗褪色试剂,用于活细胞成像(赛默飞世尔科技,目录号:P36975)
    注意:
    1. 储存在2-8°C,短期储存,≤-20°C,长期储存。
    2. 根据制造商的说明,在2-8°C下储存时,请在30天内使用本产品。在≤-20°C下储存时,产品可稳定至少六个月,最多可进行四次冻融循环。
  13. Glutamax-I(赛默飞世尔科技,目录号:35050061)
  14. 活细胞成像解决方案(见食谱)
  15. 含有血清的组织培养基(见食谱)

设备

  1. 空气置换微量移液器
    1. Starlab ErgoOne ®100-1000μl单通道移液器(目录号:S7110-1000)或Gilson F123602 PIPETMAN Classic Pipet P1000(Fisher Scientific,目录号:10387322)
    2. Starlab ErgoOne ®20-200μl单通道移液器(目录号:S7100-2200)或Gilson F123615 PIPETMAN Classic Pipet P100(Fisher Scientific,目录号:10442412)
    3. Starlab ErgoOne ®2-20μl单通道移液器(目录号:S7100-0220)或Starlab ErgoOne ®0.1-2.5μl单通道移液器(目录号:S7100) -0125)或Gilson F144801 PIPETMAN Classic Pipet P2(Fisher Scientific,目录号:10635313)或Gilson F123600 PIPETMAN Classic Pipet P20(Fisher Scientific,目录号:10082012)
  2. 基本细胞培养技术和无菌操作设备,即
    1. 生物安全柜(例如,Thermo Scientific Holten Safe 2010 Model 1.2,目录号:8207071100)
    2. 加湿培养箱设定温度为37°C(Panasonic Incusafe,目录号:MCO-20AIC)
    注意:生物安全柜的任何制造商和型号都可以,但所选的生物安全柜需要适合于容纳细胞和病毒。处理传染性病毒时,请使用适当的设施,实践和程序。请参阅当地和国际实验室生物安全指南。&nbsp;
  3. -20°C冰柜(Labcold,目录号:RLCF1520)
  4. 水浴(例如,Clifton,目录号:NE2-8D;但是,可以使用任何设定为37°C的水浴)
  5. 台式微量离心机,4°C至室温(RT),最高速度≥12,000 x g (Eppendorf,目录号:5424R)
    注意:用于旋转在长期储存SNAP探针期间可能形成的沉淀物。
  6. 活细胞成像显微镜用于长期成像。例如,尼康Ti2-E倒置宽视场显微镜(对于类似的设置,请联系您当地的尼康代表,为显微镜系统创建定制订单)
    1. 尼康Ti2-E倒置显微镜支架
    2. 电动载物台(标准Ti2编码电动XY载物台)
    3. Lumencor Spectra X LED光源
    4. CFI计划Apo Lambda 60x油/ 1.4 NA目标
    5. Photometric Prime 95B sCMOS单色相机
    6. 用于尼康Ti2-E的Semrock 32 mm滤光片(绿色):带有Ex 466/40单波段带通滤光片的滤光片(产品代码:FF01-466 / 40-25),DM495二向色分光镜(产品代码:FF495-) Di03-25x36),BA525 / 50单频带通滤波器(产品代码:FF03-525 / 50-25)
    7. 用于尼康Ti2-E的三叶草32毫米滤光片(红色):Cy3-4040C带有Ex 531/40单波段带通滤光片的滤光片立方体(产品代码:FF01-531 / 40-25),二向色镜DM 562(产品代码:FF562 -Di03-25x36,阻隔滤波器:BA 593/40(产品代码:FF01-593 / 40-25)
    8. 用于Nikon Ti2-E的Semrock 32 mm过滤器(远红色):Cy5-4040C带Ex 628/40单带带通滤波器的过滤器立方体(产品编号:FF02-628 / 40-25),单带发射器(产品编号: FF01-692 / 40-25),单波段二向色(产品编号:FF660-Di02-25x36)&nbsp;
    9. Okolab stage顶级培养箱,目录号:H301-NIKON-NZ100 / 200/500-N。设置为37°C,5%CO 2 (任何可以安装在Nikon Ti2电动载台上的制造商和型号都可以)
    10. 完美对焦系统(PFS)
    注意:
    1. 以上列表并非详尽无遗,而是列出了在创建协议1时有用的基本组件。请咨询当地的尼康代表,了解定制显微镜系统的其余组件。
    2. 我们更喜欢在带有盖玻片的玻璃底部的开放式组织培养皿中成像细胞,这需要倒置的显微镜支架。&nbsp;
    3. 由于方案1是标记蛋白长期成像的有效方法,因此保持细胞聚焦并纠正因热和机械条件引起的焦点漂移非常重要。这种活细胞成像系统包括尼康的第四代完美聚焦系统(PFS),它可以监控碟形玻璃底部和上方液体介质之间界面上的低功率红外激光束的部分反射。此功能提供连续和实时的焦点漂移校正。&nbsp;
    4. 一个大的(25 mm x 25 mm)视场(FOV)可以提高数据吞吐量并捕获超出正常FOV的其他细胞。&nbsp;
    5. 我们更喜欢基于激光照明的LED光源。 LED照明限制了光漂白和光毒性;在获取五维数据集(5-D:随着时间推移的多色3-D Z-stack)时,“温和”成像是必不可少的。&nbsp;
    6. 电动显微镜载物台和在一次实验中记录多个位置的能力进一步提高了成像系统的吞吐量。

软件

  1. 尼康NIS Elements AR成像软件
    尼康NIS Elements AR成像软件控制显微镜的所有组件。使用NIS元素设置用于采集5-D数据集的参数。用户还可以在NIS Elements中创建渲染视频,并保存5-D数据的单个帧。对于我们的宽视场图像,我们还使用Richardson-Lucy算法(设置为10次迭代)对Elements软件中的数据集进行反卷积。

程序

已发布的标签程序构成了此处描述的协议的基础(Bodor et al。,2012)。我们在此协议的基础上,还描述了一种活细胞成像装置,该装置适用于五维蛋白质周转的长期检查(即,3-D多色荧光显微镜随时间推移)。

注意:执行所有涉及生物安全柜内活细胞的液体处理步骤。只能使用无菌移液器,血清移液管,微量移液管,微管和吸头。

  1. 生产稳定的CHIKV细胞
    我们以前使用过HuH-7肝癌细胞系来获得稳定含有修饰的CHIKV复制子的细胞;我们设计了这个复制子来编码CHIKV nsP3和SNAP标签的融合蛋白(Remenyi et al。,2018)。我们将在该方案的其余部分中将来自该二级细胞系的细胞称为“稳定的CHIKV细胞”。这些细胞也组成型表达绿色荧光蛋白ZsGreen。&nbsp;
    1. 使用标准分子克隆方法生成N-,C-末端或内部SNAP-标签融合。在我们的实验系统中,我们将SNAP标签插入CHIKV nsP3的C末端区域,并在N-末端和C-末端连接处添加了一个灵活的甘氨酸氨基酸接头(Remenyi et al。>,2017)。编码SNAP f ®的质粒,一种SNAP标签蛋白,可从NEB(N9183S;或作为SNAP-Cell ®入门套件,E9100S)。
    2. 在将SNAP序列插入病毒基因组后,评估修饰的病毒或复制子是否仍然存活以及标记的蛋白质是否可以与未标记的蛋白质发挥相同的生物学功能。验证方法将根据病毒和感兴趣的蛋白质而有所不同。
    3. 以优选的组织培养形式维持细胞:我们常规地将稳定的CHIKV细胞传递到10-cm培养皿中。&nbsp;
    4. 我们用稳定的CHIKV细胞系开发了这个方案,我们从HuH-7细胞中获得了这种细胞系。支持非细胞毒性CHIKV复制子延长复制的其他细胞系包括C2C12(小鼠成肌细胞)细胞系(Remenyi et al。,2018)和BHK-21(幼仓鼠肾)细胞系(Utt) et al。,2015)。但是,我们尚未在这些细胞系中验证协议1-3。&nbsp;
  2. 通过添加足够的胰蛋白酶来覆盖附着于相应培养容器表面的细胞(例如,用于10-cm培养皿的1-2ml胰蛋白酶),从生长表面分离稳定的CHIKV细胞,并且将种子放入带有盖玻片底部的35毫米培养皿中。准备第二道菜和第三道菜,包含阳性和阴性对照。
    1. 作为阴性对照,我们在第二道菜中播种幼稚细胞(已知不表达SNAP标签蛋白)。&nbsp;
    2. NEB的SNAP细胞入门试剂盒还含有阳性对照质粒(pSNAP f -Cox8A对照质粒),可将其转染到细胞中以产生具有良好表征的线粒体定位的SNAP标记的细胞色素c氧化酶。因此,第三培养皿可含有用pSNAP f -Cox8A对照质粒转染的细胞或编码具有充分表征的亚细胞定位的SNAP标记蛋白的任何质粒。&nbsp;
    注意:
    1. 对于初学者,我们建议在本协议的标签阶段,一次只处理三个培养皿(一个培养皿用于样品,一个培养皿用于阴性对照,一个培养皿用于阳性对照)。需要更高吞吐量的高级用户可以考虑使用具有四个隔间的35mm成像盘( 例如 ,ibidiμ-Dish 35mm Quad,目录号80416)而不是使用单独的盘子。在菜肴的细分中进行同步实验( 例如 ,两个样本条件,一个阴性对照和一个阳性对照)。&nbsp;
    2. 解离贴壁细胞所需的胰蛋白酶的确切数量取决于细胞类型和细胞年龄。
    3. 我们根据所需追踪时间的长度和我们用于实验的细胞系的重复时间来调整细胞接种密度。
    4. 我们通常的目标是60%到80%的融合。例如,假设24小时的倍增时间和24小时的期望追踪期,我们将SNAP标记的细胞系播种至20%汇合,第二天用SNAP试剂染色(=脉冲,40%汇合) 24小时追逐期间的图像(允许细胞在活细胞成像期间达到80%融合)。
  3. 在标准生长条件下孵育细胞(即,37°C,5%CO 2 )过夜。&nbsp;
  4. 第二天,从-20°C冰箱中取出SNAP-Cell ® 647 SiR的冷冻储备液,在室温下解冻。&nbsp;
  5. 在1.5毫升微管中,稀释解冻的SNAP-Cell ? 647 SiR;在完全细胞培养基中进行1:1,000(终浓度0.6μM)稀释。最终体积应至少为0.6毫升,以覆盖盖玻片上方的碟形区域。用户可以将体积增加到1毫升,以降低细胞干燥的风险。短暂涡旋(≥5秒)或上下吸取稀释的标签溶液(十次)。&nbsp;
    1. SNAP-Cell ®探针是细胞可渗透的。我们的方案标记了CHIKV nsP3和SNAP标签的融合蛋白。该融合蛋白定位于细胞内区室,因此我们仅用细胞渗透性探针染色。
    2. 来自NEB的其他探针包括SNAP-Cell ® 505(绿色荧光),SNAP-Cell ® Oregon Green(绿色荧光)和SNAP-Cell ® TMR-Star(红色荧光)。优化不同SNAP标记的蛋白质和细胞系的最终浓度;蛋白质丰度和非特异性结合等因素可能因标记的蛋白质或细胞系而异。&nbsp;
    3. 根据制造商的说法,最佳底物浓度范围为1至20μM,通常在1至5μM浓度范围内获得最佳结果。我们发现即使0.6μM的SNAP-Cell ® SiR也能提供足够的染色。我们没有准备超过我们预期在一小时内消费的媒体。在优化标记条件时,始终包括阴性对照(已知未表达SNAP标签蛋白的幼稚细胞)。
      注意:三道菜的配方(在步骤7中使用0.6毫升,相应缩放以获得更多菜肴): 1998μl完整的细胞培养基; 2μlSNAP-Cell ® 647 SiR。
  6. 以最大速度(≥12,000 x g )旋转稀释的标记溶液5分钟,以去除可能的不溶性荧光碎片。去除上清液(可能看不见)时,注意不要打扰沉淀。
  7. 用0.6-1ml SNAP-标记标记培养基(在水浴中预热至37℃)替换稳定CHIKV细胞上的培养基。在37°C孵育15分钟,5%CO 2 。
    注意:在此步骤中,SNAP-Cell ® 647 SiR底物上的苄基将与SNAP标签共价连接并释放鸟嘌呤。我们发现15分钟的孵育给了我们最佳的标记。根据制造商的说法,最佳反应时间分别为5到30分钟,具体取决于实验条件和SNAP标记蛋白的表达水平。&nbsp;
  8. 在这15分钟的潜伏期内,准备一个含有冰和水的冷水浴。从-20°C冰箱中取出冷冻的ProLong Live试剂等分试样,并将等分试样在冷水浴中解冻。解冻或使用试剂时,不要超过37°C。我们将试剂保持在冰浴中,直到步骤10为止。&nbsp;
  9. 用2ml含有血清的组织培养基(预热至37℃)洗涤细胞三次。&nbsp;
  10. 用2 ml新鲜活细胞成像溶液替换常规细胞培养基,该溶液由FluoroBrite DMEM,补充胎牛血清(终浓度为10%),HEPES,Glutamax-I,非必需氨基酸和ProLong Live组成。 Antifade Reagent(参见食谱)。
  11. 最后一次洗涤后将细胞放回培养箱中。再孵育30分钟,37°C,5%CO 2 。
    注意:
    1. 此步骤的主要目的是减少SNAP试剂的背景染色。一些SNAP探针的背景染色,例如SNAP细胞 TMR-Star在某些细胞系中可能存在问题,而观察到较少的背景染色在其他人(科尔,2014年)。如果背景染色是个问题,我们建议缩短标记时间,探针浓度或增加洗涤次数。步骤10还开始与ProLong Live试剂孵育。
    2. 制造商对ProLong Live试剂的说明建议将细胞在黑暗中孵育15分钟至2小时。
    3. 根据我们的经验,当样品盘到达显微镜时,已经设置了最终成像设置,并且开始实际图像采集,含有ProLong Live的培养基的细胞总培养时间将至少为1.5小时(在步骤10-12中处理三个盘子约40分钟,从组织培养设备到显微镜处理器皿约20分钟,以及设置成像条件约30分钟。&nbsp;
  12. 用常规细胞培养基洗涤步骤9中所述的细胞。用2 ml活细胞成像培养基替换培养基(在步骤10中制备)。
    注意:如果活细胞成像不超过24小时,我们会在此步骤中使用ProLong Live解决方案补充活细胞成像介质。制造商不建议将ProLong Live解决方案留在活细胞上超过24小时。我们还计算了从该步骤完成开始的追逐期,因为它标志着荧光底物可以标记SNAP标记蛋白的最后时间点。
  13. 可选:用红色荧光或橙色荧光细胞染料染色细胞(例如,MitoTracker TM 橙色CMTMRos)。&nbsp;
    1. 在“活细胞成像缓冲液”中将1 mM MitoTracker TM 原液稀释至最终工作浓度(25-500 nM)。&nbsp;
    2. 从培养皿中取出培养基,加入含有MitoTracker TM 探针的预热(37°C)染色溶液。
    3. 将培养皿放回加湿的培养箱中并在37℃下用5%CO 2 孵育15-45分钟。
    4. 孵育期结束后,用新鲜预热的“活细胞成像缓冲液”替换染色液。&nbsp;
  14. 使用Nikon Ti2-E系统将三个培养皿(样品,阳性对照和阴性对照)转移到显微镜区域进行活细胞成像。 SNAP标记细胞的活细胞成像设置类似于活细胞荧光成像的标准配置。&nbsp;
    1. 我们建议尼康的“活细胞成像”资源,以便在适当的显微镜上进行介绍设置间隔拍摄成像。
    2. 有关其他资源,请联系您当地的尼康代表,了解“高级采集”模式下的NIS Elements培训讲义(即,多通道,多点,间隔拍摄和Z-stack)
    3. 有关其他活细胞成像设置,请参阅Bodor 等(2012)。&nbsp;
    4. 我们使用宽场成像设置来扩展相同视场的成像。我们使用尼康Ti2-E系统获得了高质量的结果。&nbsp;
    5. 有几个因素决定了我们对这个系统的偏好,即(i)Ti2-E配备了独特的完美聚焦系统(PFS),可在长时间成像过程中实时自动校正焦点漂移(ii)使用LED灯成像与基于激光的共聚焦系统相比,光源可以实现更温和的成像(iii)由于更快的器件移动和图像采集,可快速采集多点Z-堆叠(iv)快速采集可减少整体光照和随后的光毒性(v)Ti2 -E提供大视场(FOV),其捕获一个FOV内的大量单元,并且(vi)多点采集进一步增加系统的吞吐量。
  15. 具有首选成像系统的图像细胞
    1. SNAP Cell ® 647-SiR的激发最大值为645 nm,最大发射波长为661 nm。&nbsp;
    2. 使用Nikon Ti2-E倒置显微镜,我们使用Cy5染料的标准过滤器设置。稳定的CHIKV细胞也内源性地表达绿色荧光ZsGreen报告蛋白,其具有493nm的激发最大值和505nm处的发射峰(具有标准GFP滤光片组的图像)。&nbsp;
    3. 使用远红SNAP Cell ® 647-SiR的优势在于使用红色荧光细胞染料进行额外标记(例如,MitoTracker TM 橙色)和Cy3染料的过滤器设置成像是可能的。图2显示了来自游戏中时光倒流系列的代表性图像,其中我们将显微镜设置为每15分钟采集Z-堆叠,总共24小时。


      图2. 5-D成像和脉冲追踪实验的组合 A.我们仅显示来自多位置游戏中时光倒流系列的选定帧,其中显微镜每隔15分钟在8处获得Z-堆叠位置。在这个设置中,每个Z-堆栈(由41个切片组成)在45秒内完成,而花费6.5分钟获得8个位置。左侧的图像显示所有具有伪色的通道(绿色:ZsGreen,黄色:MitoTracker TM 橙色,品红色:SNAP-nsP3)。注意,标记为0小时的粒状结构仍然存在于3.75小时。这种持续存在表明这些结构保持稳定数小时。 B.从相同的游戏中时光倒流系列中选择的帧,这次在体积视图中显示。左侧的图像显示所有具有伪色的通道(绿色:ZsGreen,黄色:MitoTracker TM 橙色,品红色:SNAP-nsP3)。&nbsp;

食谱

  1. 活细胞成像解决方案(足以在步骤10,12和可选步骤13中为3个35毫米培养皿添加2毫升溶液)
    2.7毫升胎牛血清
    270μlHEPES
    270μlGlutamax-I
    270μlMEMNonessential Amino Acids
    270-540μlProLongLive Antifade Reagent
    22.96-23.22 ml FluoroBrite DMEM
    活细胞成像溶液的总体积:27毫升
  2. 含有血清的组织培养基
    500毫升Dulbecco的改良Eagle's培养基
    56.5毫升胎牛血清
    5.6毫升MEM非必需氨基酸溶液
    5.6毫升HEPES

方案2:淬火脉冲追踪实验与蔡司LSM 880 Airyscan显微镜配对

材料和试剂

  1. 微量移液器吸头,血清移液器,移液器辅助设备和用于液体处理的微管
    1. TipOne ®1,000μlXL过滤器吸头(目录号:S1122-1830)
    2. TipOne ®200μl过滤嘴(目录号:S1120-8810)
    3. TipOne ®20μl过滤嘴(目录号:S1120-1810)或SARSTEDT移液器吸头10μl(目录号:70.1130.600)
    4. Fisherbrand TM 5 ml血清移液管(目录号:13-676-10H)
    5. Fisherbrand TM 10 ml血清移液管(目录号:13-676-10J)
    6. Fisherbrand TM 25 ml血清移液管(目录号:13-676-10K)
    7. Drummond Pipet-Aid XL(目录号:4-000-205)
    8. Microtube 1.5 ml(SARSTEDT,目录号:72.690.001)
    9. 15 ml Centrifuge Tubes,Conical,Sterile(Starlab,目录号:E1415-0200)
  2. 载玻片(学院,目录号:N / A142)
  3. 滤纸(Whatman ®,目录号:1001025)
  4. 24孔板(CytoOne ®,目录号:CC7682-7524)
  5. 清洁,无菌13毫米盖玻片(#1.5)(学院,目录号:NPS16 / 1818)
  6. Parafilm ® M(Bemis,目录号:PM-996)
  7. 铝箔(Caterwrap,目录号:AKL-300-030M)
  8. 10厘米的菜肴
  9. 稳定的CHIKV细胞(见方案1)
  10. 我们的细胞系(用于基础组织培养)的特异性试剂,我们从肝癌细胞系HuH-7中获得:
    1. Dulbecco改良的Eagle's培养基(Sigma,目录号:D6429-500ML)
    2. 100%胎牛血清(Gibco,目录号:10500-064)
    3. Gibco MEM非必需氨基酸溶液(100x),自生产之日起至4个月内储存至24个月(Thermo Fisher Scientific,目录号:11140050)
    4. Gibco HEPES(N-2-羟乙基哌嗪-N-2-乙磺酸)缓冲液(100x),自生产之日起在4°C储存至24个月(Thermo Fisher Scientific,目录号:15630130)
  11. 胰蛋白酶EDTA(Sigma,目录号:T3924-500ML)
  12. PBS,磷酸盐缓冲盐水(VWR Lifescience,目录号:E404-100TABS)
  13. 用于免疫标记SNAP标记蛋白的抗体
    注意:我们使用内部生产的兔抗体检测CHIKV nsP3(Remenyi 等 ,2017和2018)。&nbsp;
  14. 山羊抗兔IgG(H + L)交叉吸收二抗,DyLight ® 405(Thermo Fisher Scientific,Invitrogen,目录号:35551)
    注意:Dylight ® 405,ZsGreen和TMR-Star的组合可为三色成像提供有效的色彩分离和灵敏度
  15. SNAP-Cell ® TMR-Star(New England Biolabs,目录号:S9105S;或作为SNAP-Cell ®入门套件的一部分,目录号:E9100S)
    注意:按照SNAP-Cell ® 647-SiR的描述准备原液。
  16. SNAP-Cell ®嵌段(溴代苯基硫脲,BTP,New England Biolabs,目录号:S9106S;或作为SNAP-Cell ®入门试剂盒的一部分,目录号:E9100S) 。储存:-20°C至少三年干燥或三个月作为溶解在DMSO中的储备溶液
  17. 固定的4%甲醛在磷酸盐缓冲盐水(PBS)中,pH 6.9
    注意:有关如何准备固定剂的详细协议,请参阅: https://www.rndsystems.com/resources/protocols/protocol-making-4-formaldehyde-solution-pbs 。简而言之,从多聚甲醛粉末(Fisher Chemical,目录号:T353-500)制备固定剂。将多聚甲醛粉末溶于1x无菌PBS(由片剂制成)(VWR Lifescience,目录号:E404-100TABS)中。用稀盐酸(HCl)(Fisher Chemical,目录号:H / 1100 / PB17)将pH调节至6.9。对于长期储存,将15毫升等分试样保存在-20°C。需要时,解冻等分试样(彻底涡旋以除去任何沉淀物)并用于每个实验。将任何剩余的溶液在4°C下保存1个月。
  18. 安装介质(ProLong Diamond Antifade Mountant,目录号:P36965)
    注意:我们使用ProLong Diamond,因为它可以保护两种荧光染料(在我们的设置中:SiR + DyLight ® 405)和荧光蛋白(在我们的设置中:ZsGreen)褪色。
  19. 完整的细胞培养基(见食谱)

设备

  1. 微量吸管
    1. Starlab ErgoOne ®100-1,000μl单通道移液器(目录号:S7110-1000)或Gilson F123602 PIPETMAN Classic Pipet P1000(Fisher Scientific,目录号:10387322)
    2. Starlab ErgoOne ®20-200μl单通道移液器(目录号:S7100-2200)或Gilson F123615 PIPETMAN Classic Pipet P100(Fisher Scientific,目录号:10442412)
    3. Starlab ErgoOne ®2-20μl单通道移液器(目录号:S7100-0220)或Starlab ErgoOne ®0.1-2.5μl单通道移液器(目录号:S7100) -0125)或Gilson F144801 PIPETMAN Classic Pipet P2(Fisher Scientific,目录号:10635313)或Gilson F123600 PIPETMAN Classic Pipet P20(Fisher Scientific,目录号:10082012)
  2. 金属镊子,带有用于提升和处理玻璃盖玻片的精细尖端(EMS,目录号:78316-1)
  3. 基本细胞培养技术和无菌操作设备,即,
    1. 生物安全柜(例如,Thermo Scientific Holten Safe 2010 Model 1.2,目录号:8207071100)
    2. 加湿培养箱设定温度为37°C(Panasonic Incusafe,目录号:MCO-20AIC)
    注意:生物安全柜的任何制造商和型号都可以,但所选的生物安全柜需要适合于容纳细胞和病毒。处理传染性病毒时,请使用适当的设施,实践和程序。请参阅当地和国际实验室生物安全指南。
  4. -20°C冰柜(Labcold,目录号:RLCF1520)
  5. 水浴(例如,Clifton,目录号:NE2-8D;但是,可以使用任何设定为37°C的水浴)
  6. 台式微量离心机,4°C至室温(RT),最大速度≥12,000 x g (Eppendorf,型号:5424R)
    注意:用于旋转在长期储存SNAP探针期间可能形成的沉淀物。
  7. 安全眼镜(保护用户免受甲醛飞溅的任何制造商或型号)
  8. 共聚焦激光扫描显微镜。例如,定制的ZEISS LSM 880系统包括以下基本组件(对于类似的设置,请联系您当地的蔡司代表以获取确切的订购信息,因为产品代码可能因客户和国家/地区而异):
    1. Axio Imager Z2支架,电动(直立系统),产品编号:430000-9902-000
    2. 电动平台,扫描平台130x85 STEP,产品编号:432033-9902-00
    3. 安装框架160x116 f / Slides 76x26,产品编号:432315-0000-000
    4. 扫描模块LSM 880,产品编号:000000-1994-956
    5. 支持f / scan模块LSM(Imager Tube),产品编号:000000-1265-660
    6. 步进电机控制f,2轴SMC2009,产品编号:432929-9011-000
    7. 实时控制器标准,产品编号:000000-2031-918
    8. Objective C PApo 63x / 1.4 Oil DIC UV-IR,产品编号:421782-9900-799
    9. 488 Vis Laser,激光氩多线25 mW,产品编号:000000-2086-081
    10. 561 Vis Laser,激光561nm用于LSM 710,产品编号:00000-1410-117
    11. 633 Vis Laser,Laser Rack LSM 880 incl。 633激光,产品编号:000000-2085-478
    12. 用于LSM的Airyscan SR模块GaAsP,产品编号:000000-2058-580
    13. 用于Airyscan的排放过滤器BP495-550 + LP570,产品编号:000000-2070-488&nbsp;
    14. 用于Airyscan的发射滤波器BP570-620 + LP645,产品编号:000000-2070-489
    15. 适用于Airyscan的双BP 420-480 + BP495-620,产品编号:000000-2095-049
    16. 用于Airyscan的双BP 465-505 + LP525,产品编号:000000-2095-051
    17. 用于Airyscan的双BP 420-480 + LP605,产品编号:000000-2095-052&nbsp;
    18. 用户PC高级Z55A高端,产品编号:000000-2142-968
    19. 'Airyscan Fast'照明模块升级为1x LSM 880系统
    注意:
    1. 以上列表并非详尽无遗,但仅列出了我们发现对我们的协议有用的基本组件。请向蔡司咨询有关完成显微镜系统的其他组件( 例如 ,舞台操纵杆,硬件许可证密钥,分束器,切换镜和物镜转换器)。&nbsp;
    2. 按照制造商的建议,在ZEN显微镜软件中应用所有必要的Airyscan设置。我们的LSM 880 Airyscan显微镜可以为480 nm的荧光团提供140 nm的最大横向分辨率和400 nm的轴向分辨率(使用ZEN Black软件处理的图像)。使用ZEN Blue软件,分辨率可以进一步提高到120 nm XY和350 nm Z分辨率。&nbsp;
    3. 我们的原始系统(带有Airyscan的LSM 880)追溯升级为'Airyscan Fast'系统,而今天的系统可以从一开始就使用'Airyscan Fast'模块进行定制。 'Airyscan Fast'照明系统允许同时同时照射4个像素,以实现样品的非常快速和温和的成像,包括以超高分辨率扫描的选项,在XY和&amp ;; Z,成像速度高达19张/秒,512 x 512像素,每秒27张图像,480 x 480,每秒6张图像,1024 x 1024。

软件

  1. ZEISS ZEN软件(用于FAST Airyscan的ZEN 2.3系统HWL)
    注意:ZEISS ZEN软件驱动LSM 880系统的所有组件,包括Airyscan成像的设置。 ZEN软件还可以获取和处理原始超分辨率图像数据集;将处理后的图像文件保存为“.czi”文件,可以将其导出到生物成像分析软件( 例如 ,Icy软件)。以此文件格式保存可确保保留与每个成像实验相关的所有元数据。&nbsp;
  2. 冰冷的生物成像软件
    注意:生物成像分析软件。我们建议使用免费软件 Icy 进行可视化,注释并量化生物成像数据集,可以从ZEISS ZEN软件包导入(de Chaumont 等 ,2012)。我们发现Icy具有直观的用户界面。

程序

我们使用已发布的标记协议(Bodor et al。,2012)来分析SNAP标记的病毒蛋白(SNAP-)的'新'(即,新翻译的)库。 NSP3)。通过将标记方法与敏感检测方法ZEISS LSM 880 Airyscan显微镜结合起来,我们可以看到“新”蛋白质和“新”蛋白质积累的细胞内位点的重新出现。在第一步(称为“猝灭”)中,非荧光SNAP-底物与实验开始时存在的SNAP-nsP3库共价结合(图3,图)。在给定量的时间(追踪)之后,第二荧光底物(脉冲)如方案1(图3,图中)中所述标记细胞。脉冲仅染色在追踪期间合成的蛋白质库。因此,ZEISS LSM 880 Airyscan显微镜只能使这个“新”(即,新翻译)池可见。 SNAP-nsP3(脉冲池+猝灭池)的总池可以用标准免疫荧光测定方法染色,其显示猝灭池,否则其将保持未被检测到(图3)。

注意:在生物安全柜内进行所有涉及活细胞或含甲醛孔的液体处理步骤。在使用活细胞时,只能使用无菌移液器,移液管,微量移液器,微管和吸头。处理甲醛溶液时请戴上安全眼镜。

  1. 将无菌盖玻片放入24孔板的不同孔中。
    注意:
    1. 根据“追逐后”时间点的数量调整井的总数。还包括一个阳性对照( 例如 ,一个没有任何SNAP细胞的 ® 阻止添加)和阴性对照( 例如 ,没有任何追逐期的井)。&nbsp;
    2. 在此协议中:两个追后时间点(3小时,6小时)和两个对照(总共四个井)。
  2. 通过添加足够的胰蛋白酶溶液(在水浴中预热至37°C)从生长表面分离稳定的CHIKV细胞,以覆盖附着于相应培养容器表面的细胞(例如,用于10-cm培养皿的1-2ml胰蛋白酶溶液)和来自步骤1的孔中的种子稳定的CHIKV细胞。
    接种密度将再次取决于实验的总持续时间和细胞的倍增时间。我们的目标是在固定0小时,3小时和6小时样品时约80%融合。因此,与0小时井相比,我们还在3小时和6小时井中播种更少的细胞,以解释更长的孵化时间(3小时井减少10-15%,6小时减少20-30%) - 哦)。
  3. 在37℃,5%CO 2 孵育过夜。
  4. 第二天,从-20°C冰箱中取出SNAP Cell ® Block的冷冻储备液,在室温下解冻。在1.5 ml微量管中,在完全培养基中稀释SNAP Cell ® Block至终浓度为2μM。短暂涡旋(≥5秒)。准备&gt;每个盖玻片200μl。在一小时内使用稀释的试剂。
  5. 用200μlSNAPCell ®替换细胞上的培养基每孔加入稀释溶液(在水浴中预热至37°C)(总共3个孔)。不要改变第四口井的介质(这将作为'积极控制')。孵育30分钟。&nbsp;
  6. 用1ml完全培养基洗涤细胞三次(在水浴中预热至37℃;这洗掉任何游离底物,这将干扰下游应用)。最后一次清洗步骤后取出介质。
  7. 向每个孔中加入1ml完全培养基(在水浴中预热至37℃)并将细胞置于组织培养箱中30分钟。
  8. 按照步骤6清洗细胞。为防止细胞变干,请勿在最后一次清洗步骤后取出清洗介质。&nbsp;
  9. 准备一个新的24孔板,并在两个孔中加入1毫升4%甲醛固定剂。&nbsp;
  10. 使用镊子将“淬火”盖玻片和“未阻塞”盖玻片之一转移到新板上并浸没在甲醛中(确保细胞层保持一侧)。 “淬火”盖玻片上的固定细胞将作为“无追踪”控制。 “未阻断的”盖玻片上的细胞将作为SNAP-试剂染色的阳性对照。
  11. 将包含剩余盖玻片的原板放回组织培养箱中。孵育细胞所需的追踪时间为3小时。&nbsp;
  12. 将装有固定盖玻片的新板置于4°C储存,直至收集到所有追逐期的所有盖玻片。在储存期间密封板的边缘以避免过度蒸发(Parafilm M的条带很好地工作)。

注意:如果淬火确实完成,则在“脉冲”期间不应发生标记。如果Airyscan显微镜仍能检测到未淬灭的SNAP标记蛋白,则表明可用的SNAP细胞 ® 阻断试剂未完全淬灭前现有游泳池。用增加浓度的SNAP细胞 阻断或延长孵育时间重复实验,直到实验条件导致SNAP标记蛋白完全淬灭

  1. 三小时后,如步骤10所述,将一个盖玻片转移到含甲醛的孔中(将板从4℃储存中取出并向第三个孔中加入1ml 4%甲醛)。这个盖玻片上的固定细胞将作为“3小时追逐”样本。&nbsp;
  2. 将包含剩余盖玻片的原板放回组织培养箱中。将细胞再培养3小时。将固定的“无追逐”和“3小时追逐”盖玻片的平板放回4°C进行储存。
  3. 重复步骤13和14(将盖玻片转移到第四个含甲醛的孔中)。最后一个盖玻片上的固定细胞将作为“6小时追逐”样本。在室温下孵育30分钟或在4℃孵育1-2小时以完成'6小时追踪'样品的固定过程。
  4. 在生物安全柜中,用PBS清洗'No chase','No quench','3-h chase'和'6-h chase'三次以去除甲醛固定剂。
    注意:此时,用户还可以选择在生物安全柜外处理24孔板,因为细胞已经经过化学固定。但是,我们仍然倾向于在生物安全柜内执行步骤17,19和20,以避免污染储备溶液和细胞培养基。
  5. 从-20°C冰箱中取出SNAP Cell ® TMR-Star的冷冻储备液,在室温下解冻。在1.5毫升微管中,稀释SNAP-Cell ® TMR-Star;在完全细胞培养基中进行1:600(终浓度1μM)稀释。每孔至少准备200μl以覆盖整个盖玻片。短暂涡旋(≥5秒)或上下吸取稀释的标签溶液(十次)。
    注意:&nbsp;
    1. 此协议还应与SNAP-Cell ® 647 SiR兼容。使用与方案1中所述相同的标记条件( 即 ,1:1,000稀释,15分钟孵育)。
    2. 24孔板中4个盖玻片的配方,根据孔数进行扩展:
      998.4μl完整细胞培养基
      1.6μlSNAP-Cell ® TMR-Star
  6. 以最大速度旋转稀释的标记溶液5分钟(≥12,000 x g )以去除可能的不溶性荧光碎片。去除上清液(可能看不见)时,注意不要打扰沉淀。
  7. 用200μlSNAP-标记标记培养基替换稳定CHIKV细胞上的培养基。在37°C,5%CO2下孵育15分钟。在步骤20-23(例如,用铝箔包裹24孔板)中保护样品免受过度曝光。
    注意:我们发现15分钟的孵育给了我们最佳的标记。根据制造商的说法,最佳反应时间分别为5至30分钟,具体取决于实验条件和SNAP标记蛋白的表达水平。
  8. 用1ml含有血清的组织培养基(预热至37℃)洗涤细胞三次。
  9. 使用优选的免疫荧光测定方案染色总标记蛋白质库。此时,在我们的案例中,用户可以处理生物安全柜关键试剂外的24孔板:针对nsP3的兔抗血清和染料偶联二抗的一抗(抗兔DyLight ® 405) &NBSP;
  10. 将免疫标记的盖玻片安装在首选的封固剂中(例如,ProLong Diamond Antifade Mountant):
    1. 将一滴固定剂放在载玻片上,用镊子从24孔板上取下免疫染色的盖玻片,然后慢慢将盖玻片(细胞侧朝下)放到液滴上。&nbsp;
    2. 使用纸巾或滤纸擦去多余的封固剂。我们通常在标准载玻片上安装两到三个盖玻片。
  11. 允许封固剂在室温和黑暗中固化(过夜或更长时间)。我们将固化的载玻片储存在4°C。&nbsp;
  12. 图片采用蔡司LSM 880 Airyscan显微镜
    1. SNAP Cell ® TMR-Star的激发最大值为554 nm,最大发射波长为580 nm。使用蓝色,绿色和红色通道设置三色成像。有关ZEISS LSM 880 Airyscan显微镜的设置,请参阅ZEN软件手册。
    2. 完成图像采集和处理后,我们使用Icy生物成像软件打开保存和处理的Airyscan数据,我们以.czi文件格式保存。图3显示了来自三色成像实验的代表性图像,包括每个通道的Icy直方图查看器的屏幕截图。


    图3.追踪期后的Airyscan显微镜显示未阻断的病毒蛋白库。来自猝灭脉冲追踪实验的代表性图像。注意,总nsP3库(用抗nsP3抗体染色)和新的nsP3库(用TMR-Star染色)的共分布表明新旧SNAP-nsP3库缺乏空间分离。我们在蓝色,绿色和红色通道中设置了用于三色成像的显微镜。我们通过拖动直方图查看器的可调边界(用箭头标记)来调整Icy平台内的图像对比度,这增强了所选通道中的对比度而不改变数据。我们选择了一个可在3小时和6小时时间点为SNAP-nsP3通道提供最佳对比度的可视范围。我们还使用相同的观察范围来显示正负控制的数据('直方图范围'窗口,标有星号*,视图范围最小值为0,最大值为8469.0像素强度值)。请注意,通过应用色彩映射'Fire'(在Icy软件中),我们可以更好地显示低强度粒状结构。&nbsp;

食谱

  1. 完整的细胞培养基
    500毫升Dulbecco的改良Eagle's培养基
    56.5毫升胎牛血清
    5.6毫升MEM非必需氨基酸溶液
    5.6毫升HEPES

方案3:用ZEISS LSM 880 Airyscan显微镜跟踪细胞裂解液中SNAP标记的病毒蛋白组装

材料和试剂

  1. 微量移液器吸头,血清移液器,移液器辅助设备和用于液体处理的微管:
    1. TipOne ®1,000μlXL过滤器吸头(目录号:S1122-1830)
    2. TipOne ®200μl过滤嘴(目录号:S1120-8810)
    3. TipOne ®20μl过滤嘴(目录号:S1120-1810)或SARSTEDT移液器吸头10μl(目录号:70.1130.600)
    4. Fisherbrand TM 5 ml血清移液管(目录号:13-676-10H)
    5. Fisherbrand TM 10 ml血清移液管(目录号:13-676-10J)
    6. Fisherbrand TM 25 ml血清移液管(目录号:13-676-10K)
    7. Drummond Pipet-Aid XL(目录号:4-000-205)
    8. 1.5毫升微管(SARSTEDT,目录号:72.690.001)
    9. 15 ml Centrifuge Tubes,Conical,Sterile(Starlab,目录号:E1415-0200)
  2. Parafilm ® M(Bemis,目录号:PM996)
  3. 铝箔(Caterwrap,目录号:AKL-300-030M)
  4. 湿纸(例如,切纸吸收滤纸,Biorad,目录号:1703965)
  5. 10厘米培养皿(Corning,目录号:430167)
  6. Ibidi双孔μ载玻片,一体式腔室载玻片,带有聚合物盖玻片和ibiTreat表面,可实现最佳细胞粘附(Ibidi,目录号:80286)
    注意:IbiTreat表面改性使聚合物盖玻片表面具有亲水性。可以使用增加盖玻片表面( ,聚赖氨酸处理)和涂层玻璃盖玻片的粘附性的替代方法(Wheeler 等。 ,2017)。&nbsp;
  7. Greiner CELLSTAR多壁培养板,6孔(Merck / Sigma Aldrich,目录号:Greiner 657160)
  8. 稳定的CHIKV细胞(见方案1)
  9. 胰蛋白酶EDTA(Sigma,目录号:T3924-500ML)
  10. 用于基础组织培养的材料和试剂(见方案1和2)
  11. SNAP-Cell ® TMR-Star(New England Biolabs,目录号:S9105S;或作为SNAP-Cell ®入门套件的一部分,目录号:E9100S),准备如SNAP-Cell ® 647-SiR所述的储备溶液
  12. PBS,磷酸盐缓冲盐水(VWR Lifescience,目录号:E404-100TABS)
  13. 4%甲醛(见方案2步骤15)
  14. Triton X-100(Sigma,目录号:T924-500ml)
  15. KCl(Fisher Chemical,目录号:P / 4240/53)
  16. NaCl(Fisher Chemical,目录号:S / 3160/60)
  17. 氯化镁(MgCl 2 )六水合物99.0-101.0%,VWR Chemicals,目录号:25108.260)
  18. 甘油(Fisher Chemical,目录号:G / 0650/17)
  19. 哌嗪-N,N'-双(2-乙磺酸)(PIPES)(Sigma,目录号:P1851-100G)
  20. Leupeptin,半硫酸盐≥85%HPLC(Sigma,目录号:L8511-5MG)
  21. Pepstatin A(Sigma,目录号:P5318-5MG)
  22. 来自牛肺的抑肽酶(Sigma,目录号:A6279-5ML)
  23. AEBSF(Pefabloc,Sigma,目录号:76307-100MG)
  24. NaOH(Fisher Chemical,目录号:S / 4920/60)
  25. 含有蛋白酶抑制剂的自制格拉斯哥裂解液(GLB)(见食谱)
    1. 1x GLB,结合原液,甘油,Triton-X和蛋白酶抑制剂(参见食谱)
    2. 该方案使用我们实验室首选的裂解缓冲液。我们还没有尝试过使用NP-40或SDS作为洗涤剂的其他裂解缓冲液。然而,请注意0.5%NP-40是应力颗粒裂解缓冲液的一个组成部分,用于制备应力颗粒核心分离的细胞裂解物(Wheeler et al。,2017)。&nbsp;
  26. GLB的库存解决方案(参见食谱)
  27. 蛋白酶抑制剂的储备溶液(见食谱)
  28. 1x GLB(见食谱)
  29. 含有血清的组织培养基(见食谱)

注意:对于#14-24,同等纯度的替代品也是合适的。

设备

  1. 微量吸管
    1. Starlab ErgoOne ®100-1,000μl单通道移液器(目录号:S7110-1000)或Gilson F123602 PIPETMAN Classic Pipet P1000(Fisher Scientific,目录号:10387322)
    2. Starlab ErgoOne ®20-200μl单通道移液器(目录号:S7100-2200)或Gilson F123615 PIPETMAN Classic Pipet P100(Fisher Scientific,目录号:10442412)
    3. Starlab ErgoOne ®2-20μl单通道移液器(目录号:S7100-0220)或Starlab ErgoOne ®0.1-2.5μl单通道移液器(目录号:S7100) -0125)或Gilson F144801 PIPETMAN Classic Pipet P2(Fisher Scientific,目录号:10635313)或Gilson F123600 PIPETMAN Classic Pipet P20(Fisher Scientific,目录号:10082012)
  2. 细胞刮刀(Fisher Scientific,目录号:08-100-241)
  3. 基本细胞培养技术和无菌操作设备,即。
    1. 生物安全柜(例如,Thermo Scientific Holten Safe 2010 Model 1.2,目录号:8207071100)&nbsp;
    2. 加湿培养箱设定温度为37°C(Panasonic Incusafe,目录号:MCO-20AIC)
    注意:生物安全柜的任何制造商和型号都可以,但所选的生物安全柜需要适合于容纳细胞和病毒。处理传染性病毒时,请使用适当的设施,实践和程序。请参阅当地和国际实验室生物安全指南。
  4. -20°C冰柜(Labcold,目录号:RLCF1520)
  5. 水浴(例如,Clifton,目录号:NE2-8D;但是,任何类型的水浴设置为37°C是合适的)
  6. 台式微量离心机,4°C至室温(RT),最大速度≥12,000 x g (Eppendorf,5424R)
    注意:用于旋转在长期储存SNAP探针期间可能形成的沉淀物。
  7. 通风橱(任何可以保护用户免受甲醛烟雾影响的制造商或型号)
  8. 共聚焦激光扫描显微镜。例如,定制的ZEISS LSM 880系统包括以下基本组件(对于类似的设置,请联系您当地的蔡司代表以获取确切的订购信息,因为产品代码可能因客户和国家/地区而异):
    1. Axio Imager Z2支架,电动(直立系统),产品编号:430000-9902-000&nbsp;
    2. 电动平台,扫描平台130x85 STEP,产品编号:432033-9902-00
    3. 安装框架160x116 f / Slides 76x26,产品编号:432315-0000-000
    4. 扫描模块LSM 880,产品编号:000000-1994-956
    5. 支持f / scan模块LSM(Imager Tube),产品编号:000000-1265-660
    6. 步进电机控制f,2轴SMC2009,产品编号:432929-9011-000
    7. 实时控制器标准,产品编号:000000-2031-918
    8. Objective C PApo 63x / 1.4 Oil DIC UV-IR,产品编号:421782-9900-799
    9. 488 Vis Laser,激光氩多线25 mW,产品编号:000000-2086-081
    10. 561 Vis Laser,激光561nm用于LSM 710,产品编号:00000-1410-117
    11. 633 Vis Laser,Laser Rack LSM 880 incl。 633激光,产品编号:000000-2085-478
    12. 用于LSM的Airyscan SR模块GaAsP,产品编号:000000-2058-580
    13. 用于Airyscan的排放过滤器BP495-550 + LP570,产品编号:000000-2070-488&nbsp;
    14. 用于Airyscan的发射滤波器BP570-620 + LP645,产品编号:000000-2070-489
    15. 适用于Airyscan的双BP 420-480 + BP495-620,产品编号:000000-2095-049
    16. 用于Airyscan的双BP 465-505 + LP525,产品编号:000000-2095-051
    17. 用于Airyscan的双BP 420-480 + LP605,产品编号:000000-2095-052&nbsp;
    18. 用户PC高级Z55A高端,产品编号:000000-2142-968
    19. 透射光检测器T-PMT,产品编号:000000-2014-999
    20. 'Airyscan Fast'照明模块升级为1x LSM 880系统&nbsp;
    注意:
    1. 以上列表并非详尽无遗,但仅列出了我们发现对我们的协议有用的基本组件。有关完成显微镜系统的其他组件( 例如 ,舞台操纵杆,硬件许可证密钥,分束器,切换镜和物镜转换器),请咨询蔡司。
    2. 按照制造商的建议,在ZEN显微镜软件中应用所有必要的Airyscan设置。我们的Airyscan显微镜可以为480 nm的荧光团提供140 nm的最大横向分辨率和400 nm的轴向分辨率(使用ZEN Black软件处理的图像)。使用ZEN Blue软件,Airyscan分辨率甚至可以进一步提高到120 nm XY和350 nm Z分辨率。&nbsp;
    3. 我们的原始系统(带有Airyscan的LSM 880)追溯升级为'Airyscan Fast'系统,而今天的系统可以从一开始就使用'Airyscan Fast'模块进行定制。 'Airyscan Fast'照明系统允许同时同时照射4个像素,以实现样品的非常快速和温和的成像,包括以超高分辨率扫描的选项,在XY和DVD中分辨率提高1.5倍。 Z,成像速度高达19张/秒,512 x 512像素,每秒27张图像,480 x 480,每秒6张图像,1024 x 1024。

软件

  1. ZEISS ZEN软件(用于FAST Airyscan的ZEN 2.3系统HWL)
    注意:ZEISS ZEN软件驱动LSM 880系统的所有组件,包括Airyscan成像的设置。 ZEN软件还可以获取和处理原始超分辨率图像数据集;将处理后的图像文件保存为“.czi”文件,可以将其导出到生物成像分析软件( 例如 ,Icy软件)。以此文件格式保存可确保保留与每个成像实验相关的所有元数据。&nbsp;
  2. 冰冷的生物成像软件
    注意:生物成像分析软件。我们建议使用免费软件 Icy 进行可视化,注释并量化生物成像数据集,可以从ZEISS ZEN软件包导入(de Chaumont 等 ,2012)。我们发现Icy具有直观的用户界面。

程序

该方案使用荧光光学显微镜来揭示在细胞裂解物中持续存在的SNAP-nsP3蛋白的稳定组装。它部分基于隔离协议(Wheeler et al。,2017),已被用于确定应力颗粒的蛋白质组和亚结构(Jain et al。,2016) )。我们的协议增加了标签(通过使用SNAP标签系统)和检测(通过使用敏感的ZEISS LSM 880 Airyscan共聚焦成像系统)的额外灵活性。

注意:执行所有涉及生物安全柜内活细胞的液体处理步骤。只能使用无菌移液器,移液管,微量移液器,微管和吸头。细胞裂解后,用户可以在生物安全柜外处理样品。处理甲醛溶液时请戴上安全眼镜。

  1. 用胰蛋白酶分离稳定的CHIKV细胞,并在6孔微量滴定板的至少一个孔中接种。此外,在至少一个孔中分离并播种不表达SNAP标记的蛋白质的幼稚细胞;这些细胞将作为阴性对照。
  2. 在标准生长条件下(即,37℃,5%CO 2 )孵育细胞过夜。
  3. 第二天,从-20°C冰箱中取出冷冻的SNAP Cell ® TMR-Star,并在室温下解冻。在1.5ml微量管中,在完全细胞培养基中稀释SNAP-Cell TMR-Star 1:600(终浓度1μM)稀释。每孔至少准备1毫升以覆盖整个区域。短暂涡旋(≥5秒)或上下吸移稀释的标记溶液(十次)。我们仅使用SNAP-Cell ® TMR-Star试剂验证了该方案。但是,该方案还应与SNAP-Cell ® 647 SiR兼容。使用与方案1中所述相同的标记条件(即,1:1,000稀释,15分钟孵育)。
    注意:两道菜的配方,相应的比例增加其他菜肴:
    &NBSP; &NBSP; &NBSP; &NBSP; &NBSP; &nbsp;1996.8μl完整细胞培养基
    &NBSP; &NBSP; &NBSP; &NBSP; &NBSP; &nbsp;3.2μlSNAP-Cell ® TMR-Star
  4. 以最大速度旋转稀释的标记溶液5分钟(≥12,000 x g )以去除可能的不溶性荧光碎片。去除上清液(可能看不见)时,注意不要打扰沉淀。
  5. 用1ml SNAP-标记标记培养基(在水浴中预热至37℃)替换稳定CHIKV细胞上的培养基。另外,更换“阴性对照”孔中的培养基。在37°C孵育15分钟,5%CO 2 。
    注意:我们发现15分钟的孵育给了我们最佳的标记。根据制造商的说法,最佳反应时间分别为5至30分钟,具体取决于实验条件和SNAP标记蛋白的表达水平。
  6. 用2ml含有血清的组织培养基(在水浴中预热至37℃)洗涤细胞三次。最后一次清洗后请勿取出介质。
  7. 将细胞放回加湿的培养箱中45-60分钟。
    我们使用延长的洗脱期(与方案1相比)进一步减少非特异性背景结合。此外,我们不会在此协议中添加ProLong Gold Live,因为活细胞成像仅限于步骤8中的可选质量控制步骤。
  8. 可选:用显微镜确认SNAP-nsP3的荧光染色
    1. 配备10倍或20倍物镜的基本宽场荧光显微镜足以通过显微镜目镜评估SNAP标记的质量。如果需要记录染色质量,请使用连接的相机拍摄照片。另外,确认含有阴性对照的孔中没有染色。&nbsp;
    2. 我们建议在第一次测试标签条件时执行此步骤。快速进行成像并尽快进入步骤9,以限制细胞健康状况的恶化。如果这在逻辑上不可行,我们建议您直接进入步骤9。
  9. 用1 ml PBS替换完整的细胞培养基,并使用单独的细胞刮刀从每个孔的生长区域分离细胞。将得到的细胞悬浮液转移到1.5ml微管中。在1,500 x g 的沉淀细胞,在室温下3分钟。去除上清液。
    可选的暂停点:如果我们想在以后进行裂解,我们将颗粒冷冻在≤-20°C。
  10. 通过添加300μl含有蛋白酶抑制剂的冰冷的格拉斯哥裂解缓冲液来裂解颗粒(参见食谱)。通过反复移液,轻弹管或涡旋确保完全重悬颗粒。如果颗粒来自冰箱,在冰上解冻5分钟,然后加入裂解缓冲液。&nbsp;
  11. Vortex裂解30秒。放在冰上30秒。我们在涡旋循环之间将样品返回到冰中以防止在室温下延长的孵育期。或者,裂解物可以在冷室中涡旋。
  12. 重复步骤11三次。
  13. 在850℃下旋转,在4℃下旋转5分钟以除去残留的细胞碎片。
    注意:在此步骤后我们不进一步纯化SNAP-nsP3的组装,而是在显微镜分析中使用粗上清液。测试在18,000 x g 的后续离心是否可以沉淀SNAP-nsP3的组件将是有趣的。这一步骤对于分离纯种群的应力颗粒核心至关重要(Wheeler 等 ,2017)。在此步骤之后,用户可以处理生物安全柜外的细胞裂解液。
  14. 将整个体积的上清液转移到双孔Ibidi腔室塑料载玻片上。丢弃沉淀的细胞碎片。
    注意:我们注意到在这一步骤上清液可能会出现浑浊。已知作为RNP组分的蛋白质经历液 - 液相变,并且浊度是由于形成小的富含蛋白质的液滴(Molliex 等。 ,2015 )。然而,我们还没有测试纯化的SNAP-nsP3是否可以在溶液中形成类似的液滴。
  15. 在4°C下在黑暗中的潮湿环境中孵育过夜。
    1. 为了获得加湿条件,我们用提供的塑料盖覆盖Ibidi载玻片,并将载玻片放入10厘米的培养皿中,该培养皿中还含有湿纸(例如,切割的Western印迹滤纸片)。&nbsp ;
    2. 我们用Parafilm M进一步密封餐具并用铝箔包裹以防止光线照射。我们一夜之间孵化,使SNAP-nsP3的组装有足够的时间沉淀到室底。&nbsp;
    3. 未来的实验可以确定SNAP-nsP3沉积到腔室载玻片底部的最短持续时间;这些信息有助于缩短完成第3号议定书所需的总时间。&nbsp;
  16. 在通风橱中,向含有细胞裂解物的孔中加入1ml 4%甲醛。在室温下孵育一小时。
    注意:用微量移液管缓慢分配甲醛。我们添加此步骤来修复SNAP-nsP3的程序集;固定可降低组件在随后的清洗步骤中从井表面脱离的可能性。
  17. 用PBS洗涤孔三次。确保在两次洗涤之间井不会变干。&nbsp;
  18. 将载玻片转移到选择的显微镜系统:
    如协议2中所述,我们使用在Airyscan模式下操作的ZEISS LSM 880成像系统。对TMR-Star使用适当的滤波器设置,其激发最大值为554 nm,最大发射波长为580 nm。此外,以明场模式获取图像。该图像可以提供所检查的生物结构的额外记录。
  19. 我们建议每个实验至少拍摄三张图片:
    我们用63x物镜获取图像。将经过处理的Airyscan文件导出到Icy生物成像软件。通过在软件直方图查看器中拖动查看范围的边界来调整Icy软件中的图像对比度(请参见图4,箭头)。我们更喜欢使用“Fire”色彩图查看SNAP-nsP3通道。使用Icy的控件放大感兴趣的区域。有关代表性图像,请参见图4。


    图4.使用Airyscan显微镜分析细胞裂解物。 我们根据方案3处理样品并检测含有TMR-Star标记的SNAP-nsP3的颗粒结构。我们选择了Icy中的“Fire”色图来显示来自TMR-Star染色的信号。我们还通过拖动直方图查看器的可调边界(用箭头标记)来调整图像对比度。我们使用63x物镜以1的缩放因子获得图像。在图像处理过程中,我们还通过增加Icy软件中的数字变焦来放大感兴趣区域(ROI)(即,ROI1为3.3倍,ROI2为20倍)。最后,我们还获得了一个明场通道,以提供参考并揭示裂解液中存在的所有造成对比的结构。

食谱

  1. 自制格拉斯哥裂解液(GLB)含蛋白酶抑制剂,5 ml体积


  2. GLB的库存解决方案
    将适量的固体加入蒸馏的H 2 O中,制备PIPES储液(用3 M NaOH调节pH至7.2),KCl,NaCl,MgCl 2 浓度列于表中。将这些储备溶液储存在4°C。
  3. 蛋白酶抑制剂的储备溶液,以下列方式储存:
    Leupeptin在-20°C时1 mg / ml
    Pepstatin A在-20°C下1 mg / ml
    抑肽酶2 mg / ml,4°C
    AEBSF(Pefabloc)在4℃下100mM
  4. 1x GLB
    根据表格结合储备溶液,甘油,Triton-X和蛋白酶抑制剂。用蒸馏的H 2 O将体积调节至5ml,用3M NaOH将pH调节至7.2
    注意:每次实验都使用新的1x GLB。
  5. 含有血清的组织培养基
    500毫升Dulbecco的改良Eagle's培养基
    56.5毫升胎牛血清
    5.6毫升MEM非必需氨基酸溶液
    5.6毫升HEPES

数据分析

我们认为显微镜用户在Nikon NIS Elements AR软件(.nd2文件格式)或ZEISS ZEN软件(.czi文件格式)中保存的未处理图像文件是“原始”数据。因此,这些协议中的数据分析仅用于定性目的,并且仅由图像可视化和所获取图像的数字处理(例如,调整亮度,对比度和伪色)组成,可用于尼康NIS Elements AR软件或Icy生物成像软件。尽管也可以从这些图像中提取定量数据,但我们的初始方法开发(侧重于新标记和成像方法的应用)并未包括这些类型的生物图像信息学方法。将严格的生物图像信息学技术纳入此处描述的协议,未来的研究可能会受益匪浅。

致谢

Wellcome Trust Investigator Award资助了这项工作(WT 096670,授予Mark Harris)。 Wellcome Trust多用户设备奖(Zeiss LSM 880仪器,WT104918MA,'用于生物医学科学的活细胞多功能成像')使共享设备的购买成为可能。资助者在研究设计,数据收集和解释方面没有任何作用,也没有决定提交出版作品。我们感谢Sally Boxall博士和利兹大学生物科学学院的生物成像设施,以获取和帮助Airyscan显微镜。作者承认Gina Gamble和Kate Lewis博士使用Nikon Ti2-E倒置显微镜提供了帮助。我们认识到Bodor 等人的先前研究(2012),其中描述了脉冲追踪和淬火 - 脉冲追逐方法。我们感谢Mark B. Carascal和Samuel Ko博士在修改稿件时提出的意见。

利益争夺

作者声明不存在利益冲突或竞争利益。

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Remenyi, R., Li, R. and Harris, M. (2019). On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates. Bio-protocol 9(4): e3177. DOI: 10.21769/BioProtoc.3177.
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