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Mar 2022

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NBD-lipid Uptake Assay for Mammalian Cell Lines
哺乳动物细胞系的 NBD 脂质摄取测定   

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Abstract

All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their substrate specificity, and their regulation remain to be elucidated. Here, we describe an optimized quantitative flow cytometry-based lipid uptake assay utilizing nitrobenzoxadiazolyl (NBD) fluorescent lipids to study lipid internalization in mammalian cell lines, which allows characterizing lipid transporter activities at the plasma membrane. This approach allows for a rapid analysis of large cell populations, thereby greatly reducing sampling variability. The protocol can be applied to study a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane, and to uncover the dynamics of lipid transport at the plasma membrane.


Graphic abstract:


Internalization of NBD-labeled lipids from the plasma membrane of CHO-K1 cells.


Keywords: Flow cytometry (流式细胞仪), Lipid transport (脂质运输), Mammalian cells (哺乳动物细胞), NBD-lipid (NBD-脂质), Plasma membrane (质膜)

Background

A remarkable feature of many biological membranes is that their phospholipids are asymmetrically distributed across the lipid bilayer, a phenomenon known as transbilayer lipid asymmetry. This lipid asymmetry is essential for several vital cellular functions, including regulation of membrane protein activity, signaling, and vesicle formation in the secretory and endocytic pathways. Thus, establishing and regulating lipid asymmetry is crucial for cells, and a number of membrane proteins have evolved to fulfill the function of cross-bilayer lipid transporters. These transporters include ATP-dependent flippases and floppases—which catalyze the inward movement of lipids from the extracellular/luminal leaflet to the cytoplasmic leaflet, and the outward movement of lipids from the intracellular leaflet to the extracellular/ luminal leaflet, respectively, and ATP-independent scramblases (Holthuis and Levine, 2005; Contreras et al., 2010). Despite their fundamental cellular importance, key aspects of how these lipid transporters operate await elucidation.


A subgroup of P-type ATPases, the P4-ATPases, emerged as a major group of lipid flippases that form heterodimeric complexes with members of the Cdc50 (cell division control 50) protein family (Lopez-Marques et al., 2014). Mutations in these transporters generate impairments in physiological processes and, in humans they have been linked to diseases such as intrahepatic cholestasis and cerebellar ataxia (van der Mark et al., 2013). While initially characterized as aminophospholipid flippases, recent studies of individual family members from yeast, parasites such as Leishmania, plants, and mammalian cells show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids, synthetic alkylphospholipids, and glycolipids (Roland et al., 2019; Shin and Takatsu, 2019).


Characterization of phospholipid movement at a quantitative level in the plasma membrane of eukaryotic cells is frequently based on fluorescent lipids with a covalently linked 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in the sn-2 position. These lipid analogs have a NBD group attached to a short fatty acid chain (C6) and maintain most of the properties of endogenous phospholipids, except that they are more water-soluble, which facilitates incorporation from the medium into the outer monolayer of the plasma membrane. Transport of these probes is usually monitored by extracting the residual fraction of analogs not transported across the membrane with bovine serum albumin (BSA). As BSA extracts all analogs from the exoplasmic monolayer of the plasma membrane, the inaccessible fraction reflects analogs that have been redistributed across the plasma membrane into cells.


The protocol presented here utilizes flow cytometry to study NBD-lipid internalization at a quantitative level in mammalian cells, exemplified on Chinese hamster ovary-K1 (CHO-K1) cells, and has been applied by us to fibroblasts (Pomorski et al., 1996), lymphocytes (Fischer et al., 2006), and myoblasts (Grifell-Junyent et al., 2022). For lipid uptake assays optimized for fungi and plants, the reader is referred to previously published protocols (Jensen et al., 2016; López-Marqués and Pomorski, 2021). The protocol includes the preparation of NBD-lipids, labelling of cells with NBD-lipids, flow cytometry measurements, and data analysis. Additionally, cells are subjected to lipid analysis via thin-layer chromatography (TLC) to assess metabolic conversion of the NBD-lipids. This protocol can be easily adapted to parasites such as Toxoplasma and Leishmania (Weingartner et al., 2011; dos Santos et al., 2013; Chen et al., 2021), and has a broad range of applications, including: i) screening of mammalian cell lines for their lipid uptake profile, ii) testing the impact of gene knockouts on lipid internalization at the plasma membrane, and iii) uncovering the dynamics of lipid transport at the plasma membrane.


Things to consider before starting

  1. Choice of NBD-lipid

    The NBD-lipids need to fulfill three important requirements: (i) they cannot be modified at their polar head group, as this is the key structural element for substrate recognition by ATP-dependent flippases and floppases (Theorin et al., 2019); (ii) they have to incorporate readily into the outer plasma membrane, to get the time zero for the uptake kinetic; and (iii) they have to be extractable by BSA. These requirements are best met by lipid analogs in which one fatty acid has been replaced by a short chain of six carbons (C6) carrying the fluorescent NBD moiety in the sn-2 position of the glycerophospholipids, or linked by an amide bond to the ceramide backbone, as well as lyso-glycerophospholipid derivates labeled at the sn-1 position (Figure 1).



    Figure 1. Chemical structure of NBD-lipids used in lipid uptake assays.

    Fluorescent analogs of phosphatidylserine with different acyl chain lengths and positions of the NBD moiety. A) 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (C16:0-C6:0 NBD-PS). In these analogs, one fatty acid has been replaced by a short chain of six carbons (C6) carrying the fluorescent NBD moiety in the sn-2 position, whereas the sn-1 chain is composed of sixteen carbons (C16). B) 1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine (C14:0-C6:0 NBD-PS); similar to the analog in (A), but the sn-1 chain is composed of fourteen carbons (C14). C) 1-NBD-dodecanoyl-2-hydroxy-sn-glycero-3-phospho-serine (NBD-lyso-PS). In these analogs, the C12 sn-1 acyl chain carries the NBD moiety. D) The NBD moiety is attached to the carbon twelve of a C18 sn-1 acyl chain, whereas the sn-2 chain is composed of four carbons (Colleau et al., 1991).

  2. Metabolic conversion of the NBD-lipids

    In nucleated cells, lipid internalization is not only affected by transbilayer movement and intracellular membrane trafficking, but also by lipid metabolism. Some lipid analogs are known to be actively metabolized by phospholipase activities at the cell surface. Examples are the hydrolysis of NBD- phosphatidic acid to NBD-diacylglycerol (Pagano and Longmuir, 1985), and the hydrolysis of NBD-sphingomyelin to NBD-ceramide, both occurring at the plasma membrane. The fluorescent analogs of diacylglycerol and ceramide undergo rapid spontaneous transbilayer movement, and therefore label intracellular membranes (Pagano and Sleight, 1985).


    Thus, the conversion of an NBD-lipid may be followed by rapid spontaneous movement of its metabolic products and should always be critically evaluated. Another frequent modification is the hydrolysis of the NBD-lipid analogs into lyso-derivates by phospholipase A2 activities, which results in the removal of the fatty acid attached to the sn-2 position. The labeled C6 fatty acids liberated are released into the medium, hampering quantitative analysis of NBD-lipid internalization. To block this conversion, the assay is typically performed in the presence of phospholipase inhibitors (Pomorski et al., 1996; Fischer et al., 2006; Grifell-Junyent et al., 2022).


    Therefore, it is highly recommended to analyze the degree of NBD-lipid conversion using, for example, lipid extraction in organic solvents followed by thin layer chromatography (see under Procedure, section D).

  3. Lipid internalization by endocytosis

    Lipid analogs inserted into the outer plasma membrane leaflet can also be internalized by endocytosis. To suppress uptake by endocytosis, the assay is typically performed at 20°C or below.

Materials and Reagents

  1. Mammalian cell culture

    In this study, we used Chinese hamster ovary-K1 cells (CHO-K1; Cell number: RCB0285, Riken BRC, Japan) kindly provided by Dr. Kentaro Hanada, that were cultured in cell culture medium (see Recipes).

    1. Sterile serological pipettes (e.g., Serological pipettes of 5 mL, 10 mL and 25 mL; Sarstedt, catalog numbers: 86.1253.001, 86.1254.001, 86.1685.001)

    2. Sterile culture vessels T-75 flasks (e.g., Sarstedt, catalog number: 83.3911)

    3. Basal cell culture medium (e.g., high glucose DMEM; Sigma-Aldrich, catalog number: D6546)

    4. Fetal bovine serum (FBS; e.g., Sigma-Aldrich, catalog number: F4135)

    5. L-glutamine stock of 200 mM (e.g., Sigma-Aldrich, catalog number: G7513)

    6. Penicillin-streptomycin (e.g., Sigma-Aldrich, catalog number: P4333)

    7. Trypsin-EDTA solution (e.g., Sigma-Aldrich, catalog number: T3924)

    8. Hanks’ Balanced Salt solution (HBSS; e.g., Sigma-Aldrich, catalog number: H6648)

    9. Trypan Blue Solution, 0.4% (Thermo Fisher Scientific, catalog number: 15250061)

    10. Tyrode's balanced salt solution (TBSS; see Recipes)


  2. Preparation of NBD-lipids

    1. C16:0-C6:0 NBD-lipids purchased in chloroform including:

      NBD-PC (Avanti Polar Lipids, catalog number: 810130)

      NBD-PE (Avanti Polar Lipids, catalog number: 810153)

      NBD-PS (Avanti Polar Lipids, catalog number: 810192)

      NBD-SM (Avanti Polar Lipids, catalog number: 810218)

    2. Dimethyl sulfoxide (DMSO; Carl Roth, catalog number: 4720.4)


  3. Labelling of cells with NBD-lipids

    1. 3-(-4-octadecyl)-benzoylacrylic acid (OBAA; Sigma-Aldrich, catalog number: SML0075)

    2. Phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, catalog number: p7626)

    Note: OBAA is a potent inhibitor of phospholipase A2 (Eintracht et al., 1998); PMSF has been reported to inhibit phospholipases, as well as some esterases (James, 1978; Estevez et al., 2012; Gadella and Harrison, 2000).


  4. Analysis of NBD-lipid uptake and metabolism

    1. PYREX® 11 mL screw cap culture tubes, 16 × 100 mm (Corning, catalog number: 9825-16), and centrifuge glasses DURAN® with conical bottoms, 12 mL (Carl Roth, catalog number: K211.1). If not using a cap (see point D2), centrifuge glasses DURAN® with round bottoms, 12 mL (Carl Roth, catalog number: C102.1) can also be used instead.

    2. Tube caps with polypropylene for centrifuge glasses (Ohemen Labor, catalog number: 6702588). Using of caps is optional.

    3. Polypropylene tubes of 15 mL and 50 mL capacity (e.g., Falcon tubes, Sarstedt, catalog numbers: 62.554.502 and 62.547.254)

    4. 1.5-mL microcentrifuge tubes (Sarstedt, catalog number: 72.690.001)

    5. Tubes for flow cytometer (e.g., Sarstedt, catalog number: 55.484)

    6. Reusable glass media bottles with cap, 250 mL and 1 L (e.g., Thermo Fisher Scientific, catalog numbers: 15456113 and 15486113)

    7. Graduated pipette serological, 5 mL (Sigma-Aldrich, catalog number: BR27112)

    8. MacromanTM pipette (Gilson, catalog number: F110120)

    9. Disposable glass Pasteur pipettes, 230 mm (VWR, catalog number: 612-1702)

    10. Glass Pasteur pipette bulb (VWR, catalog number: 470123-222)

    11. Bovine serum albumin, essentially fatty acid free (BSA; Sigma-Aldrich, catalog number: A6003)

      Note: Fatty acid-free BSA allows for efficient extraction of NBD-lipids from cellular membranes, due to the unoccupied fatty acid binding sites.

    12. Propidium iodide ≥94.0% (HPLC) (PI; Sigma-Aldrich, catalog number: P4170)

    13. Flow cytometer cleaning solution (e.g., Sysmex, catalog number: 04-4009_R)

    14. Flow cytometer sheath fluid (e.g., Sysmex, catalog number: 04-4007_R)

    15. Chloroform 99-99.4% ethanol-stabilized and certified for absence of phosgene and HCl (VWR, catalog number: 22711.290)

    16. Ethanol absolute ≥99.8% (VWR, catalog number: 20821.321)

    17. Methanol ≥99.8% (VWR, catalog number: 20847)

    18. Triethylamine (Carl Roth, catalog number: X875.3)

    19. Thin layer chromatography (TLC) Silica gel 60, 10 × 20 cm (Merck, Darmstadt, Germany, catalog number: 1.05626.0001)


  5. Media and buffers

    1. Cell culture medium (see Recipes)

    2. TBSS buffer (see Recipes)

    3. NBD-lipid stocks (see Recipes)

    4. PMSF stock of 200 mM (see Recipes)

    5. OBAA stock of 5 mM (see Recipes)

    6. BSA (20% w/v) in TBSS (see Recipes)

    7. PI stock of 1 mg mL-1 (see Recipes)

    8. Alkaline running buffer (see Recipes)

Equipment

  1. Analytical balance (e.g., Sartorius Entris-i II, 220 g/0.1 mg, Buch Holm, catalog number: 4669128)

  2. Eppendorf Research® plus pipettes P20, P200, P1000 (Eppendorf, catalog numbers: 3123000039, 3123000055, 3123000063)

  3. Pipette tips 10 µL, 200 µL, 1,000 µL (Sarstedt, catalog numbers: 70.760.002, 70.3030.020, 70.3050.020)

  4. Neubauer counting chamber (improved Dark lines, 0.1 mm) and cover glasses (20 mm × 26 mm × 0.4 mm)

  5. Flow cabinet to work with organic solvents

  6. Biological safety cabinet certified for handling of biological materials (e.g., Herasafe KSP Class II Biological Safety Cabinets, Thermo Fisher Scientific)

  7. Hamilton 700 Series Syringes 25 µL, 100 µL, 1,000 µL (Hamilton Company, Nevada, USA)

  8. Centrifuge with rotor for 15 mL and 50 mL polypropylene tubes (e.g., Eppendorf 5810 R; Wesseling, Germany)

  9. Autoclave sterilizer (e.g., Systec VX-65, Systec, Linden, Germany)

  10. Water distillation system

  11. Incubator with humidity and gas control to maintain 37°C and 95% humidity in an atmosphere of 5% CO2 in air (e.g., Binder, Tuttlingen, Germany)

  12. Inverted light microscope equipped with a 10× objective (HI PLAN I 10×/0.22 PH1; Leica DMi1, Mannheim, Germany)

  13. Water bath (e.g., WPE45 Memmert, Schwabach, Germany) for mammalian cells and for NBD-lipid labelling (Julabo CORIO C-BT5, catalog number: 9011305)

  14. Vortex mixer (e.g., Vortex Genie 2 Scientific Industries Inc., catalog number: SI-0236)

  15. Vacuum Pump V-100 with Interface I-100 (Buchi, catalog numbers: 11593636 and 11593655D)

  16. Glass desiccator Boro 3.3 with socket in lid, 20 cm, including stopcock (BRAND GmbH, catalog number: 65238)

  17. Tubing (BRAND GmbH, catalog number: 143275)

  18. Developing chamber for TLC (Roth Carl, catalog number 3133.1)

  19. Freezers at -20°C and -80°C

  20. Refrigerator

  21. Fluorescence Imager

    For this protocol, a Chemidoc MP Imaging System (Bio-Rad, Munich, Germany) with a 530/28 filter and light Blue Epi illumination was used.

  22. Flow cytometer

    For this protocol, a CyFlow® SL flow cytometer (Sysmex Partec, Münster, Germany) was used, equipped with a solid-state laser of 488 nm.

  23. Computer with monitor (e.g., DELL U2415)

Software

  1. FloMax® software (Sysmex Partec)

  2. FlowJoTM v10.0.7 Software (FlowJo)

  3. Image LabTM software (Bio-Rad)

Procedure

When utilizing cell lines that grow in suspension, skip Steps A1–A5 and start at Step A6.

  1. Preparation of mammalian cells

    1. Grow adherent cells in sterile culture vessels (T-75 flask) in appropriate cell culture medium in a tissue culture incubator (37°C, 5% CO2, 95% humidity) until they reach a confluency of 70–80%.

      Note: Given that for each time point and NBD-lipid, at least ~104 cells are required, a total of 3 × 106 cells were collected per lipid tested, which corresponded to three T-75 flask of CHO-K1 cells at a confluency of 70–80%. For other cell lines, growth conditions and confluency degrees might require adjustment.

    2. Remove media and wash cells twice with 5 mL of HBSS (Ca2+ and Mg2+ free, pre-warmed at 37°C).

    3. Add 1.5 mL of trypsin-EDTA solution (pre-warmed at 37°C) and incubate the T-75 flasks in a tissue culture incubator (37°C, 5% CO2, 95% humidity).

    4. After 5 min, check under the microscope if the cells have detached. If the cell line allows it, tap the flask in the side to help the detachment of the cells from the bottom.

      Note: Cells in suspension will show a round-shaped morphology.

    5. Stop trypsinization by adding 7.5 mL of appropriate cell culture medium (pre-warmed at 37°C).

      Note: For cells grown in suspension, Steps A1–A5 are omitted.

    6. Transfer the cell suspension into a 50 mL Falcon and set aside 50 µL of this in a 1.5-mL microcentrifuge tube for cell counting, e.g., using the hemocytometer (see below).

    7. Centrifuge cells in the 50 mL Falcon tube at 500 × g and room temperature for 10 min and discard the supernatant.

    8. Add 10 mL of TBSS buffer (see Recipes) at room temperature to the cell pellet, and resuspend completely by pipetting up and down.

      Note: This additional washing step ensures the removal of phenol red, a pH indicator present in DMEM media that interferes with the NBD-lipid fluorescence quantification.

    9. Centrifuge the cells in the 50 mL Falcon tube at 500 × g and room temperature for 10 min and discard the supernatant.

    10. Resuspend the cells in TBSS buffer (see Recipes) to a final concentration of ~106 cells mL-1.

    11. To block the conversion of NBD-lipids by cellular phospholipases, add PMSF and OBAA to a final concentration of 1 mM and 5 µM, respectively, to the cell suspension.

    12. Gently mix and incubate at 20°C in a water bath placed in a cold room for 10 min. The assay is typically performed at 20°C or below to suppress endocytosis.


  2. Cell counting

    1. Prepare the hemocytometer by cleaning the chambers and coverslip with isopropanol. Dry the hemocytometer by using lint-free tissue. Place the glass cover slip over the counting chambers.

    2. Add 50 µL of 0.4% trypan blue stock solution to 50 µL of cell suspension (Step A6) to obtain a 1:1 dilution.

    3. Load the hemocytometer with 10 µL of cell suspension and examine immediately under an inverted phase contrast microscope at low magnification.

    4. Count the number of viable (seen as bright cells) and non-viable cells (stained blue) in the large outer quadrants.

    5. Calculate the percentage of viable cells: % viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100. Cell viability should be at least 95%.

    6. Calculate the cell concentration, based on the premise that each square accounts for a volume of 10-4 mL of cell suspension.

    7. To obtain the total number of viable cells per ml of aliquot, multiply the total number of viable cells by 2 (the dilution factor for trypan blue) and the correction factor of 104 (volume of each square).


  3. NBD-lipid uptake assay

    Note: In order to prevent nonspecific binding of the NBD-lipids, all steps must be performed in glass tubes.

    1. Prior to the start of the assay, prepare round bottom glass tubes with NBD-lipid stocks in DMSO (see Recipes) and 1.5-mL microcentrifuge tubes on ice: one empty and one with 30 µL of 20% BSA (see Recipes) for each time point.

      Note: We routinely use a final BSA concentration of 4.6% (w/v) for NBD-lipid extraction. However, the amount of BSA required for extraction, as well as the incubation time, may vary depending on cell type and lipid analog used in the assay (Fellmann et al., 2000). To determine the optimal conditions, label the cells at 4°C and measure the cell-associated fluorescence after different time of contact of the cell suspension with BSA.

    2. To start cell labeling, add 3 mL of the cell suspension (from Step A12) into the glass tubes containing the NBD-lipid stocks, and gently vortex for 2 s (Figure 2).

    3. Incubate cells with NBD-lipids for the desired time periods, e.g., 0, 5, 10, 20, 30, 45 and 60 min. Note that for t=0 min, samples need to be collected immediately after addition of cells to the lipid suspensions. When analysing uptake of several NBD-lipids in parallel, start cell additions spaced by 1 min intervals.

    4. At each time point, gently vortex the cell suspensions for 2 s using low vortex power settings to avoid pelleting of the cells, and take two aliquots of 100 µL. Transfer one aliquot to a pre-cooled empty 1.5-mL microcentrifuge tube, and the other aliquot to a precooled 1.5-mL microcentrifuge tube containing 30 µL of 20% BSA (see Figure 2).

      Note: At this point, additional samples can be taken for analyzing metabolic conversion of the fluorescent lipid analogs using lipid extraction and thin layer chromatography analysis (see section D).

    5. Keep the samples on ice for a maximum of one hour after collecting the last sample, and analyse them via flow cytometry.

    6. Turn on the CyFlow® SL flow cytometer, start the computer, and open the FlowMax software.

    7. Click on the panel “Instrument Settings” and manually enter the parameters (Table 1).

      Note: Save the employed parameters on the instrument to reload in future runs.

    8. Open the gating menu, select “Polygon” and click on “New”. Generate a gate in the plot, by default termed R1, with the parameters forward scatter (FSC) in the x-axis and side scatter in the y-axis (Dawaliby et al., 2016), covering most of the surface in the center of the plot.

      Note: Gates can be saved and loaded in future runs.

    9. Click on the option “Setup” to open the Setup window. Select as the maximal count number for R1 10,000 cells. Click “OK”.

      Note: We typically count cells until reaching 10,000 in gate R1 (corresponding to population P1).

    10. To rinse the machine before use, add 1 mL of TBSS into a flow cytometer tube and run for about 1 min at a speed between 4–8 µL s1.

      Note: Make sure there are very little to no counts and thus, no contamination or residual cells in the flow cytometer. In case of contaminants, rinse for longer times with TBSS and, if still persistent, wash with the cleaning solution, and then again with TBSS.

    11. Set the flow speed at 3–4 µL s-1 for counting on average 300 cells s-1. Speed can be adjusted if necessary.

    12. Transfer the cell sample into a flow cytometer tube, followed by addition of 1 µL of 1 mg mL-1 PI and 1 mL of TBSS. Shortly vortex and place the sample tube in the sample holder. PI labelling allows excluding dead cells from the analysis, which readily absorb NBD-lipids because of the disruption of their plasma membranes. Upon addition of a new cell sample, a message appears to save the data just collected. In the equipment used, samples are automatically saved in the .fcs format (check “Data analysis” on how to proceed for samples analysis).



      Figure 2. Schematic illustration of the NBD-lipid uptake assay.

      For labeling, cells (106 cells mL-1) pre-incubated with phospholipase inhibitors at 20°C are transferred to glass tubes containing the DMSO-dissolved NBD-lipid, and then incubated for different time periods. At each time point, two aliquots of 100 µL are taken. One aliquot is transferred to a pre-cooled empty 1.5-mL microcentrifuge tube, and the other aliquot to a precooled 1.5-mL microcentrifuge tube containing 30 µL of 20% BSA. Samples are subsequently analysed by flow cytometry. Details on the individual steps are described in the text.


      Table 1. Parameters employed for flow cytometry analysis of the mammalian cell line CHO-K1 labelled with NBD-lipids. Settings employed for each parameter, including gain, scale (Log), lower limit (L-L) and upper limit (U-L). FSC, forward scatter; SSC, side scatter; FL1, channel for NBD fluorescence; FL3, channel for PI fluorescence. The flow cytometer used is equipped with a blue solid state laser (488 nm, 20 mW). FL1 and FL3 fluorescence are recorded with a band pass filter IBP 527/30 and a long pass filter RG 630 nm, respectively. Note that these values might require adjustment depending on the cell line and/or flow cytometer employed.

      Parameter Gain Log L-L U-L
      FSC 170 lin 110 999.9
      SSC 190 lin 10 999.9
      FL1 220 Log4 10 999.9
      FL3 415 Log4 10 735.9


  4. Analysis of fluorescent lipid metabolism

    Lipid extraction can be performed based on the modified (Bligh and Dyer, 1959) protocol. The method is based on the partitioning of lipids in a biphasic mixture of chloroform and methanol. Methanol disrupts hydrogen bonds between lipids and proteins following addition of an organic solvent such as chloroform. Except centrifugation, all steps described are performed in a flow cabinet to avoid direct exposure to organic solvents.

    1. Transfer aliquots of the cell suspension at the given time points to 12 mL glass tubes. We typically analyse the metabolism after 60 min, by taking volumes corresponding to ~4 nmol NBD-lipid (150 µL of cells labelled with NBD-PE; 300 µL of cells labelled with NBD-PC; 600 µL of cells labelled with NBD-PS or NBD-SM).

    2. Add up to 2 mL of ddH2O considering the volume already present in each glass tube.

    3. Add 2.2 mL of methanol followed by 2 mL of chloroform using glass graduated pipettes and vortex carefully for 3–5 s to achieve a single phase.

    4. Centrifuge the tubes at 500 × g for 5 min to obtain two separated phases.

    5. Transfer the lower phase (chloroform phase containing lipids) into a conical bottom glass tube using a glass Pasteur pipette.

    6. For a second round of extraction, add 1 mL of chloroform to the original tubes, vortex shortly and centrifuge again at 500 × g for 5 min.

    7. Transfer the lower phase into the glass tubes from Step D5.

    8. Prepare four conical bottom glasses and add 4 nmol of NBD-PC, NBD-PE, NBD-PS and NBD-SM resuspended in chloroform:methanol (1:1, v/v) from the same lipid stocks used to label the cells. These samples will serve as standards to compare to the extracted lipid samples.

    9. Dry both the lipid extracts and the standard samples under vacuum at 250 mbar overnight or under a gentle stream of nitrogen gas for 30 min to 1 h.

    10. On a TLC silica plate, mark 1 cm lines as loading spots with a pencil. These should be 1.5 cm apart from the bottom edge and 1 cm apart from the side edges and from each other. Draw a total of eight lines, four for the NBD-lipid standards and four for the extracted NBD-lipid samples (Figure 3A). Samples information can be written by pencil under the loading spots. Do not use excessive force when writing on a TLC plate as this will remove the silica coating.

    11. Carefully pour the running buffer (see Recipes) into the developing chamber until filled to ~0.5 cm of heigh. Close with the lid to allow air saturation for ~20 min.

    12. Dissolve the extracted dried lipids (~4 nmol) and the 4 nmol NBD-lipid standards in a volume of ~5 µL of chloroform/methanol (1:1, v/v).

    13. Apply the dissolved NBD-lipids onto the TLC silica plate using a glass Pasteur pipette, by placing it carefully on top of the 1 cm loading spot to avoid peeling off the silica and ensuring even distribution. Let the organic solvent evaporate before adding more sample on top on the loading spot.

    14. Place the TLC plate as evenly as possible in the chamber with a slight tilt. Close the lid and run for 20–25 min, or until the running front has reached a distance of 1 cm from the top edge.

    15. Remove the TLC plate from the chamber, mark the running front with a pencil, and let it dry for 15–30 min.

    16. Image NBD-lipid fluorescence in the Chemidoc Imaging System, using the Image LabTM software and emission filter 530/28 nm under Blue light Epi illumination (Figure 5C).

      Note: Place the TLC plate on top of a transparent plastic bag to avoid scratching the Chemidoc tray surface.

    17. Optional: Band intensities can be quantified using the Image LabTM software, e.g., to assess the percentage of metabolic conversion of lipids. Low hydrolysis (<10%) does not affect the NBD-lipid internalization kinetics. However, for higher hydrolysis (>10%) it is crucial to determine whether the converison occurs intracellularly or on the cell surface, to interpret the results.



      Figure 3. Preparation of the TLC.

      A) Marking of the loading spots on the silica plate with a pencil and loading of the NBD-lipids with a glass Pasteur pipette (for details see text). The lanes should not be placed too close to the edge: keep a distance of 1.5 cm as indicated by red arrows. B) Silica plate placed into the TLC chamber containing the alkaline running buffer and covered with the glass lid. The plate is developed until the solvent is about half a centimeter below the top of the plate.

Data analysis

  1. Export data in original format (.fcs) from the flow cytometer and import into the FlowJo sofware.

  2. Double click on top of the first file. A plot will open and cells can be visualized as dots. Select FCS and SSC for the x and y-axis respectively, and selecte the logarithmic scale. Adjust the scale range.

  3. Generate a gate around the cell population and label it as P1, referring to CHO-K1 cells in this case (Figure 4A). Double click on top of the selected population, another panel plot will open.

  4. Select FL3 (PI fluorescence) for the x-axis and keep SSC for the y-axis in the new plot. Generate a gate around the cell population with low PI-fluorescence, which includes only alive cells, and label as P2 (Figure 4B).

  5. Double click on top of the P2 population. A new plot will open. Select FL1 (NBD-lipid fluorescence) for the x-axis and SSC (side scatter) for the y-axis (Figure 4C).

  6. In the FlowJo interface, the populations P1 and P2 created appear below the .fcs file. Right-click on each and select “Copy analysis to group” to apply gates to all the other files.

  7. Check that the generated gates fit for all samples, and move to fit if necessary.

  8. In the FlowJo interface, click on Workplace and then click in Add Statistics. Select “Geometrical Mean”, population P2, and parameter FL1.

  9. Save analysed samples as Worspace (WSP) files and as Excel (xls) files.

  10. Calculate the percentage of NBD-lipid that cells have uptaken at each timepoint (Figure 5A, B). The geometrical mean of each timepoint of the sample without BSA addition is considered as the 100% NBD-fluorescence. The geometrical mean value of the sample with BSA at a given time point is the relative percentage compared to the value assigned to the 100% (from the sample without BSA; Eq. 1). The new calculated percentage is referred to as non-extractable NBD-lipid in step 11 and Eq. 1 or as NBD-lipid uptake in Figure 5B and elsewhere. Plot the resulting percentages of internalized NBD-lipid at each time point (Figures 5A and 5B).



  11. Fit data according to the equation for a single-exponential curve (e.g., using Microsoft Excel and the add-in programm Solver):



    where

    y is the fitted value for NBD-lipid fluorescence in percentage (%) at each time point,

    t is the time after NBD-lipid addition,

    a is the percentage of non-extractable NBD-lipid at t=0,

    b is the percentage of non-extractable NBD-lipid at steady state,

    c is the rate coefficient.



    Figure 4. Gating cell populations using the FlowJo software.

    Cells were labelled with NBD-lipids and collected at a given time point. This data illustrates the gating procedure corresponding to cells labelled for 60 min and treated with BSA. A) Cells were visualized in a logarithmic scale plot of side- and forward-scattering. A population P1 was defined as corresponding to mammalian cells and excluding other possible particles (e.g., cell debris). B) The P1 population was visualized in a new panel plotting side scattering and red propidium iodide (PI; dos Santos et al., 2013)-fluorescence. A population P2 was defined by selecting alive cells and excluding dead cells, labelled with PI. C) The P2 population was subsequently plotted in a side-scatter and green NBD-fluorescent panel, thus showing alive cells that internalized NBD-lipids.



    Figure 5. Exemplary time lapse of NBD-lipid uptake in CHO-K1 cells and lipid metabolism.

    Cells incubated with the indicated NBD-lipids at 20°C were analysed by flow cytometry after back-extraction to BSA. A) Histogram of NBD-PC fluorescence intensity in CHO-K1 cells after BSA incubation measured at the indicated timepoints. Note that a corresponding histogram for cells without BSA treatment will show a constantly high cell-associated fluorescence over time, provided that there is no metabolic modification, e.g., hydrolysis of the NBD-lipid analogs into lyso-derivates and free NBD-fatty acid. B) Time course of NBD-lipids internalization in CHO-K1 cells, shown as percentage of fluorescence intensity relative to fluorescence from cells treated in the absence of BSA. Plotted lines represent the best fit to a single-exponential curve. C) Thin layer chromatogram of NBD-lipids extracted from CHO-K1 cells after 60 min incubation and after BSA treatment. Lipid samples were resolved onto a silica plate along with standards and imaged for fluorescence. Diacylglycerol (DAG) ceramide (Cer) and NBD-X [6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid] (hexanoic acid), products of NBD-lipids metabolic conversion are also indicated. The asterisk (*) indicates either unidentified contaminants or lipid species derived from NBD-PE. The arrow on the right shows the running direction of the alkaline buffer. The running origin (O) and running front (F) are marked. NBD, nitrobenzoxadiazol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin.

Recipes

Buffers were prepared using double distilled water (ddH2O), which was obtained using an in-house water distillation system. Alternatively, all buffers are prepared using ultrapure water with purification sensitivity of 18 MΩ cm-1 at 25°C.

  1. Cell culture medium

    Open a 500 mL flask of high-glucose DMEM medium

    Add 50 mL of fetal bovine serum

    Add 5 mL of L-glutamine

    Add 5 mL of penicillin-streptomycin

    Prepare in sterile cabinet; store at 4°C

  2. TBSS buffer

    136 mM NaCl

    2.6 mM KCl

    1.8 mM CaCl2

    1 mM MgCl2

    0.36 mM NaH2PO4

    5.56 mM glucose

    5 mM HEPES

    Adjust pH to 7.4 with NaOH. Complete volume to 1 L. Sterilize by filtering using a 0.22 µm filter.

    Store at 4°C for up to several months.

  3. NBD-lipid stocks

    All steps must be performed in glass tubes in order to prevent nonspecific binding of lipids.

    Note: Chloroform is a chemical hazard. Do not breathe gas/fumes/vapor/spray. Wear suitable protective clothing. Work in a fume hood.

    1. Use a glass syringe to transfer the desired amount of NBD-lipid into a disposable 12 mm-diameter glass tube. Typically, we label 3 mL of cell suspension with 80 nmol of NBD-PE, 40 nmol of NBD-PC, and 20 nmol of NBD-PS or NBD-SM.

      Note: These amounts have been optimized to allow for using the same flow cytometry settings when analysing the labeled cells.

    2. Dry the lipids under a 250 mbar vacuum overnight or under a gentle stream of nitrogen gas for 30 min to 1 h, so that a dried lipid film is formed at the bottom of the tube.

    3. Resuspend the NBD-lipids in 20 µL of DMSO shortly before use. Add the DMSO in a circular fashion, by placing the piepette tip onto the walls of the glass tube and on top of the dried lipid film. Slowly pipette up and down repeating the circular movement until all the lipid film is resuspended.

    Note: DMSO lipid suspensions are prone to precipitation owing their hygroscopic nature and should be freshly prepared. DMSO is a chemical hazard. Wear suitable protective clothing.

  4. PMSF stock of 200 mM

    Weight 34.838 mg with protective gear to avoid inhalation or contact with skin.

    Dissolve in 1 mL ethanol.

    Store at -20°C.

    Note: PMSF is a chemical hazard, specially in its solid state, highly toxic if swalled, and causing severe skin burns and eye damage upon contact. Wear suitable protective clothing and work in a fume hood when preparing the stock solution. Handle the stock solution carefully.

  5. OBAA stock of 5 mM

    Weight 2.14 mg

    Add 1 mL of absolut ethanol

    Store at -80°C

    Note: Wear suitable protective clothing.

  6. BSA (essentail fatty acid-free, 20% w/v) in TBSS

    Weight 200 mg BSA in a 15 mL Falcon tube

    Add 1 mL of TBSS

    Incubate at 37°C in a water bath until partially dissolved.

    Store in the fridge until next day and use within one week; do not freeze the solution.

  7. PI stock of 1 mg mL-1

    Weight 1 mg of PI

    Add 1 mL of ddH2O

    Store at -20°C

    Note: PI is a suspected carcinogen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations. Wear suitable protective clothing.

  8. Alkaline running buffer

    Mix the following volumes in 30:35:35:7 v/v/v/v (mL) chloroform:ethanol:triethylamine: ddH2O in a blue-cap glass.

    Shake vigourously. Prepare on the same day of the experiment.

    Note: For the size of the TLC chamber (W × D × H: 235 × 116 × 220 mm) described in this protocol, the alkaline running buffer was prepared by mixing 15:17.5:17.5:3.5 v/v/v/v (mL) chloroform:ethanol:triethylamine: ddH2O. The organic solvents are chemical hazard. Do not breathe gas/fumes/vapor/spray. Wear suitable protective clothing. Work in a fume hood.

Acknowledgments

This protocol was adapted from our previous work (Pomorski et al., 1996; Weingartner et al., 2011; dos Santos et al., 2013; Grifell-Junyent et al., 2022). The work was supported by the Lundbeck Foundation (R221-2016-1005 to T.G.P) and the German Research Foundation (GU 1133/11-1 to T.G.P). M.G.J. acknowledges funding from the University of Copenhagen and the Research Internship Exchange Program of the Ruhr University Bochum. We gratefully acknowledge Dr. Marcus Peters for granting us access to the flow cytometer.

Competing interests

The authors declare that no competing interests exist.

References

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简介

[摘要] 所有真核细胞都配备了跨膜脂质转运蛋白,它们是膜脂质不对称、囊泡运输和膜融合的关键参与者。这些转运蛋白的突变与人类疾病之间的联系凸显了它们在细胞稳态中的重要作用。然而,它们的活动、底物特异性和调节的许多关键特征仍有待阐明。在这里,我们描述了一种优化的基于定量流式细胞仪的脂质摄取测定,利用硝基苯并恶二唑(NBD)荧光脂质来研究哺乳动物细胞系中的脂质内化,从而可以表征质膜上的脂质转运蛋白活性。这种方法可以快速分析大细胞群,从而大大降低采样变异性。该协议可用于研究广泛的哺乳动物细胞系,测试基因敲除对质膜脂质内化的影响,并揭示质膜脂质转运的动力学。

图形摘要:


来自 CHO-K1 细胞质膜的 NBD 标记脂质的内化。

[背景] 许多生物膜的一个显着特征是它们的磷脂不对称地分布在脂质双层中,这种现象称为跨双层脂质不对称。这种脂质不对称对于几种重要的细胞功能至关重要,包括在分泌和内吞途径中调节膜蛋白活性、信号传导和囊泡形成。因此,建立和调节脂质不对称性对细胞至关重要,并且已经进化出许多膜蛋白来实现跨双层脂质转运蛋白的功能。 这些转运蛋白包括 ATP 依赖性翻转酶和翻转酶——它们分别催化脂质从细胞外/腔小叶向内运动到细胞质小叶,以及脂质从细胞内小叶向外运动到细胞外/腔小叶, 和 ATP 非依赖性加扰酶(Holthuis 和 Levine,2005;Contreras等人,2010) 。尽管它们具有基本的细胞重要性,但这些脂质转运蛋白如何运作的关键方面仍有待阐明。
P 型 ATP 酶的一个亚组,即 P4-ATP 酶,作为脂质翻转酶的主要组出现,与 Cdc50(细胞分裂控制 50)蛋白家族的成员形成异二聚体复合物(Lopez-Marques等人,2014) 。这些转运蛋白的突变会导致生理过程受损,并且在人类中,它们与肝内胆汁淤积和小脑共济失调等疾病有关(van der Mark et al. , 2013) 。虽然最初被描述为氨基磷脂 最近对来自酵母、利什曼原虫等寄生虫、植物和哺乳动物细胞的个体家族成员的研究表明, P4-ATP 酶的底物特异性不同,并介导更广泛的脂质底物的转运,包括溶血磷脂、合成烷基磷脂和糖脂(Roland等人,2019 年;Shin 和 Takatsu,2019 年) 。
sn中共价连接的 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) 基团-2位置。这些脂质类似物具有连接到短脂肪酸链 (C6) 上的 NBD 基团,并保持内源性磷脂的大部分特性,除了它们更易溶于水,这有助于从培养基掺入到质膜的外单层中.这些探针的转运通常通过用牛血清白蛋白 (BSA) 提取未通过膜转运的类似物的残留部分来监测。 由于 BSA 从质膜的外质单层中提取所有类似物,因此不可接近的部分反映了已通过质膜重新分布到细胞中的类似物。
此处介绍的方案利用流式细胞术在哺乳动物细胞中定量研究 NBD-脂质内化,以中国仓鼠卵巢-K1 (CHO-K1) 细胞为例,并已被我们应用于成纤维细胞(Pomorski et al. , 1996 ) 、淋巴细胞 (Fischer et al. , 2006) 和成肌细胞(Grifell-Junyent et al. , 2022) 。对于针对真菌和植物优化的脂质摄取测定,读者可参考之前发布的方案(Jensen等人,2016 年;López-Marqués 和 Pomorski,2021 年) 。该协议包括制备NBD 脂质、用 NBD 脂质标记细胞、流式细胞术测量和数据分析。此外,通过薄层色谱 (TLC) 对细胞进行脂质分析,以评估 NBD-脂质的代谢转化。该方案可以很容易地适应弓形虫和利什曼原虫等寄生虫(Weingartner等人,2011;dos Santos等人,2013;Chen等人,2021) ,并具有广泛的应用,包括: i ) 筛选哺乳动物细胞系的脂质摄取谱,ii) 测试基因敲除对质膜脂质内化的影响,以及 iii) 揭示质膜脂质转运的动力学。

开始之前要考虑的事情
NBD-脂质的选择
NBD-脂质需要满足三个重要要求:( i )它们不能在其极性头部基团处进行修饰,因为这是 ATP 依赖性翻转酶和翻转酶识别底物的关键结构元素 (Theorin等人,2019) ; (ii) 它们必须容易地结合到外质膜中,以使吸收动力学的时间为零; (iii) 它们必须可由 BSA 提取。脂质类似物最能满足这些要求,其中一个脂肪酸已被短链六碳 (C6) 取代,该短链在甘油磷脂的sn-2位置携带荧光 NBD 部分,或通过酰胺键与神经酰胺骨架,以及标记在sn-1位置的溶血-甘油磷脂衍生物(图 1)。


图 1. 用于脂质摄取测定的 NBD-脂质的化学结构。
具有不同酰基链长度和 NBD 部分位置的磷脂酰丝氨酸的荧光类似物。 A ) 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl} -sn -glycero- 3-phosphoserine (C16:0-C6:0 NBD -PS)。在这些类似物中,一个脂肪酸已被一个六碳短链 (C6) 取代,该短链在sn-2位携带荧光 NBD 部分,而sn-1链由十六个碳 (C16) 组成。 B ) 1-myristoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl} -sn -glycero- 3-phosphoserine (C14:0-C6:0 NBD -PS);类似于(A) 中的类似物,但sn-1链由十四个碳 (C14) 组成。 C )1-NBD-十二烷酰基-2-羟基-sn-甘油-3-磷酸-丝氨酸(NBD-lyso- PS )。在这些类似物中,C12 sn-1酰基链带有 NBD 部分。 D ) NBD 部分连接到 C18 sn-1酰基链的碳 12 上,而sn-2链由四个碳组成(Colleau et al. , 1991) 。

NBD-脂质的代谢转化
在有核细胞中,脂质内化不仅受跨双分子层运动和细胞内膜运输的影响,还受脂质代谢的影响。已知一些脂质类似物被细胞表面的磷脂酶活性代谢。实例是 NBD-磷脂酸水解成 NBD-二酰基甘油(Pagano 和 Longmuir,1985) ,以及 NBD-鞘磷脂水解成 NBD-神经酰胺,两者都发生在质膜上。二酰基甘油和神经酰胺的荧光类似物经历快速的自发跨双层运动,因此标记细胞内膜(Pagano和Sleight,1985) 。
因此, NBD-脂质的转化可能会伴随其代谢产物的快速自发运动,因此应始终进行严格评估。另一种常见的修饰是通过磷脂酶 A 2活性将 NBD-脂质类似物水解成溶血衍生物,这导致连接到sn-2位置的脂肪酸被去除。释放的标记 C6 脂肪酸被释放到培养基中,阻碍了 NBD-脂质内化的定量分析。为了阻止这种转化,通常在存在磷脂酶抑制剂的情况下进行测定(Pomorski等人,1996;Fischer等人,2006;Grifell-Junyent等人,2022) 。
因此,强烈建议分析 NBD-脂质转化的程度,例如,在有机溶剂中提取脂质,然后进行薄层色谱(参见程序,D 部分)。

通过内吞作用进行脂质内化
插入外质膜小叶的脂质类似物也可以通过内吞作用内化。 为了抑制内吞作用的摄取,该测定通常在 20°C 或更低温度下进行。


关键字:流式细胞仪, 脂质运输, 哺乳动物细胞, NBD-脂质, 质膜

材料和试剂
哺乳动物细胞培养
Kentaro博士友情提供的中国仓鼠卵巢 K1 细胞(CHO-K1;细胞号:RCB0285,Riken BRC,日本) 花田,在细胞培养基中培养(见食谱)。
无菌血清移液器(例如,5 mL、10 mL 和 25 mL 的血清移液器; Sarstedt ,目录号: 86.1253.001、86.1254.001、86.1685.001)
无菌培养容器 T-75 烧瓶(例如, Sarstedt ,目录号:83.3911)
基础细胞培养基(例如, 高糖DMEM; Sigma-Aldrich, 目录号:D6546)
胎牛血清(FBS;例如,Sigma-Aldrich,目录号:F4135)
200 mM的L-谷氨酰胺原液(例如,Sigma-Aldrich,目录号:G7513)
青霉素-链霉素(例如,Sigma-Aldrich,目录号:P4333)
胰蛋白酶-EDTA 溶液(例如,Sigma-Aldrich,目录号:T3924)
汉克斯平衡盐溶液(HBSS;例如,Sigma-Aldrich,目录号:H6648)
台盼蓝溶液,0.4%(Thermo Fisher Scientific,目录号:15250061)
Tyrode 平衡盐溶液(TBSS;见食谱)
NBD-脂质的制备
购买的 C16:0-C6:0 NBD-脂质 在氯仿中,包括:
NBD-PC(Avanti Polar Lipids,目录号:810130)
NBD-PE(Avanti Polar Lipids,目录号:810153)
NBD-PS(Avanti Polar Lipids,目录号:810192)
NBD-SM(Avanti Polar Lipids,目录号:810218)
二甲基亚砜(DMSO;Carl Roth,目录号: 4720.4)
用 NBD 脂质标记细胞
3-(-4-十八烷基)-苯甲酰基丙烯酸(OBAA; Sigma-Aldrich ,目录号:SML0075)
苯基甲基磺酰氟(PMSF; Sigma-Aldrich,目录号:p7626 )
注意: OBAA 是一种有效的磷脂酶 A 2抑制剂 (Eintracht 等人,1998 年) ;据报道 PMSF 可抑制磷脂酶以及一些酯酶 (James,1978;Estevez 等人,2012; Gadella 和 Harrison,2000) 。
NBD-脂质摄取和代谢分析
PYREX ® 11 mL 螺旋盖培养管,16 × 100 毫米 ( Corning ,目录号:9825-16)和具有锥形底部的离心玻璃 DURAN®,12 mL(Carl Roth,目录号:K211.1)。如果不使用盖子(参见 D2 点) ,也可以使用 12 mL 圆底离心玻璃 DURAN ® (Carl Roth,目录号:C102.1)。
用于离心玻璃的聚丙烯管盖( Ohemen Labor,目录号:6702588)。使用大写字母是可选的。
容量为 15 mL 和 50 mL 的聚丙烯管(例如, Falcon 管, Sarstedt ,目录号:62.554.502 和 62.547.254)
1.5-mL 微量离心管(Sarstedt,目录号:72.690.001)
流式细胞仪管(例如, Sarstedt ,目录号:55.484)
可重复使用的带盖玻璃培养基瓶,250 mL 和 1 L(例如Thermo Fisher Scientific,目录号: 15456113 和 15486113)
刻度移液管血清学,5 mL(Sigma-Aldrich,目录号:BR27112)
Macroman TM移液器(Gilson,目录号:F110120)
一次性玻璃巴斯德移液器,230 mm(VWR,目录号:612-1702)
玻璃巴斯德移液管灯泡(VWR,目录号:470123-222)
牛血清白蛋白,基本上不含脂肪酸(BSA;Sigma-Aldrich,目录号:A6003)
注意:不含脂肪酸的 BSA 可以有效地从细胞膜中提取 NBD-脂质, 由于未占据的脂肪酸结合位点。
碘化丙啶≥ 94.0% (HPLC) (PI;Sigma-Aldrich,目录号:P4170)
流式细胞仪清洁溶液(例如, Sysmex,目录号:04-4009_R)
流式细胞仪鞘液(例如, Sysmex,目录号:04-4007_R)
氯仿 99-99.4% 乙醇稳定并证明不存在光气和 HCl(VWR,目录号:22711.290)
无水乙醇≥99 。 8%(VWR,目录号:20821.321)
甲醇≥99 。 _ 8%(VWR,目录号:20847)
三乙胺(Carl Roth,目录号:X875.3)
薄层色谱(TLC)硅胶60,10 × 20 cm(Merck,Darmstadt,Germany,目录号:1.05626.0001)
媒体和缓冲区
细胞培养基(见配方)
TBSS 缓冲液(见配方)
NBD-脂质库存(见食谱)
PMSF 200 mM 库存(见配方)
5 mM OBAA 库存(见配方)
TBSS 中的 BSA (20% w/v) (见配方)
PI 库存 1 mg mL -1 (见食谱)
碱性运行缓冲液(参见配方)
设备
分析天平(例如, Sartorius Entris-i II,220 g/0.1 mg,Buch Holm,目录号:4669128)
Eppendorf Research ® plus 移液器 P20、P200、P1000(Eppendorf,目录号:3123000039、3123000055、3123000063)
移液器吸头 10 µL、200 µL、 1、000 µL( Sarstedt ,目录号:70.760.002、70.3030.020、70.3050.020)
Neubauer 计数室(改进的暗线,0.1 mm)和盖玻片(20 mm × 26 mm × 0.4 mm)
适用于有机溶剂的流动柜
生物安全柜经认证可处理生物材料(例如Herasafe KSP Class II 生物安全柜、赛默飞世尔科技)
Hamilton 700 系列注射器 25 µL、100 µL、1,000 µL(Hamilton Company,内华达州,美国)
带转子的离心机,适用于 15 mL 和 50 mL聚丙烯管(例如, 埃彭多夫 5810 R;韦瑟林,德国)
高压灭菌器(例如, Systec VX-65,Systec,林登,德国)
水蒸馏系统
2的气氛中保持 37°C 和 95% 的湿度(例如, Binder,图特林根,德国)
配备 10 ×物镜的倒置光学显微镜 (HI PLAN I 10 × /0.22 PH1; Leica DMi1, Mannheim, Germany)
水浴(例如, WPE45 Memmert , Schwabach ,Germany),用于哺乳动物细胞和 NBD-脂质标记( Julabo CORIO C-BT5,目录号:9011305)
涡旋混合器(例如, Vortex Genie 2 Scientific Industries Inc.,目录号:SI-0236)
带接口 I-100 的真空泵 V-100( Buchi ,目录号:11593636 和 11593655D)
玻璃干燥器Boro 3.3,盖子上有插座,20 cm,包括旋塞阀(BRAND GmbH,目录号:65238)
管道(BRAND GmbH,目录号:143275)
TLC 显影室(Roth Carl,目录号 3133.1)
-20°C 和 -80°C 的冷冻箱
冰箱
荧光成像仪
对于该协议,使用了具有 530/28 滤光片和浅蓝色 Epi 照明的Chemidoc MP 成像系统(Bio-Rad,慕尼黑,德国)。
流式细胞仪
对于该协议,使用配备有 488 nm 固态激光器的CyFlow® SL流式细胞仪(Sysmex Partec ,Münster,Germany) 。 
带显示器的电脑(例如, 戴尔 U2415)
软件
FloMax ®软件 ( Sysmex Partec)
FlowJo TM v10.0.7 软件( FlowJo )
Image Lab TM软件(Bio-Rad)
程序
当使用悬浮生长的细胞系时,跳过步骤 A1 - A5 并从步骤 A6 开始。
哺乳动物细胞的制备
在组织培养箱(37°C,5% CO 2 ,95% 湿度)中,在无菌培养容器(T-75 烧瓶)中的适当细胞培养基中培养贴壁细胞,直到它们达到 70 – 80% 的融合度。
注意: 鉴于对于每个时间点和 NBD-脂质,至少需要约 10 4个细胞,每个测试的脂质共收集了 3 × 10 6 个细胞,这对应于三个 T-75 烧瓶中的 CHO-K1 细胞汇合70 – 80%。对于其他细胞系,可能需要调整生长条件和汇合度。 
2+和 Mg 2+ ,在 37°C 下预热)清洗细胞两次。
加入 1.5 mL 胰蛋白酶-EDTA 溶液(在 37°C 下预热)并在组织培养箱(37°C、5% CO 2 、95% 湿度)中孵育 T-75 烧瓶。
5 分钟后,在显微镜下检查细胞是否脱落。如果细胞系允许,轻敲烧瓶的侧面以帮助细胞从底部脱离。
注意:悬浮细胞将呈现圆形形态。
通过添加 7.5 mL 适当的细胞培养基(在 37°C 预热)停止胰蛋白酶消化。
注意:对于悬浮培养的细胞,步骤 A1 - A5 被省略。
将细胞悬液转移到 50 mL Falcon 中,并在 1.5 mL 微量离心管中留出 50 μL 用于细胞计数,例如, 使用血细胞计数器(见下文)。
× g和室温下将 50 mL Falcon 管中的细胞离心10 分钟并弃去上清液。
在室温下向细胞沉淀中加入 10 mL 的 TBSS 缓冲液(参见配方),并通过上下移液完全重新悬浮。 
笔记: 这一额外的洗涤步骤可确保去除酚红,这是 DMEM 培养基中存在的一种 pH 指示剂,会干扰 NBD-脂质荧光定量。 
× g和室温下将 50 mL Falcon 管中的细胞离心10 分钟并弃去上清液。
将 TBSS 缓冲液中的细胞重悬(参见配方)至终浓度为 ~10 6 个细胞 mL -1 。
为了阻止细胞磷脂酶对 NBD 脂质的转化,将 PMSF 和 OBAA 分别添加到细胞悬浮液中,最终浓度为 1 mM 和 5 μM。
轻轻混合并在 20°C 的水浴中放置在冷室中孵育10 分钟。该测定通常在 20°C 或更低温度下进行以抑制内吞作用。
细胞计数
通过用异丙醇清洁腔室和盖玻片来准备血细胞计数器。使用无绒组织干燥血细胞计数器。将玻璃盖玻片放在计数室上。
将 50 μL 的 0.4% 台盼蓝库存溶液添加到 50 μL 的细胞悬浮液(步骤 A6)中,以获得 1:1 的稀释。
用 10 μL 的细胞悬浮液加载血细胞计,并在低放大倍率的倒置相差显微镜下立即检查。
计算大外象限中活细胞(被视为明亮细胞)和非活细胞(染成蓝色)的数量。
计算活细胞的百分比:% 活细胞 = [1.00 - (蓝色细胞数÷总细胞数)] × 100。细胞活力应至少为 95%。
在每个正方形占 10 -4 mL 细胞悬浮液体积的前提下计算细胞浓度。
要获得每毫升等分试样的活细胞总数,请将活细胞总数乘以 2(台盼蓝的稀释因子)和 10 4的校正因子(每个正方形的体积)。
NBD-脂质摄取测定
注意:为了防止 NBD 脂质的非特异性结合,所有步骤都必须在玻璃管中进行。
在检测开始之前,准备圆底玻璃管,在 DMSO 中加入 NBD 脂质储备(参见食谱)和 1.5 mL 微量离心管在冰上:一个是空的,一个是 30 µL 的 20% BSA(参见食谱)时间点。
注意:我们通常使用 4.6% (w/v) 的最终 BSA 浓度进行 NBD-脂质提取。然而,提取所需的 BSA 量以及孵育时间可能会因测定中使用的细胞类型和脂质类似物而异(Fellmann 等人,2000) 。要确定最佳条件,请在 4°C 下标记细胞,并在细胞悬液与 BSA 接触不同时间后测量细胞相关荧光。
要开始细胞标记,请将 3 mL 的细胞悬浮液(从步骤A1 2)添加到含有 NBD-脂质库存的玻璃管中,并轻轻涡旋 2 秒(图 2)。
将细胞与 NBD-脂质一起孵育所需的时间段,例如, 0、5、10、20、30、45和 60 分钟。请注意,对于 t=0 分钟,需要在将细胞添加到脂质悬浮液后立即收集样品。在并行分析几种 NBD 脂质的吸收时,以 1 分钟的间隔开始添加细胞。
在每个时间点,使用低涡流功率设置轻轻涡旋细胞悬浮液 2 s,以避免细胞沉淀,并取两个 100 μL 的等分试样。将一个等分试样转移到预冷的 1.5-mL空微量离心管中,将另一个等分试样转移到含有 30 µL 20% BSA 的预冷 1.5-mL 微量离心管中(参见图 2)。
注意:此时,可以使用脂质提取和薄层色谱分析来分析荧光脂质类似物的代谢转化(见 D 节)。
收集最后一个样品后,将样品放在冰上最多一小时,并通过流式细胞仪对其进行分析。
打开CyFlow ® SL 流式细胞仪,启动计算机,然后打开FlowMax软件。
单击面板“仪器设置”并手动输入参数(表 1)。
注意:将使用的参数保存在仪器上,以便在以后的运行中重新加载。
打开门控菜单,选择“多边形”并单击“新建”。在图中生成一个门,默认情况下称为 R1,x 轴为前向散射 (FSC) 参数,y 轴为侧向散射(Dawaliby et al. , 2016) ,覆盖中心的大部分表面的情节。
注意: Gates 可以在以后的运行中保存和加载。
单击“设置”选项以打开“设置”窗口。选择 R1 10,000 个细胞的最大计数。单击“确定”。
注意:我们通常在门 R1 中计数细胞直到达到 10,000(对应于 P1 人口)。
要在使用前冲洗机器,请将 1 mL 的 TBSS 添加到流式细胞仪管中,并以 4–8 µL s  1之间的速度运行约 1 分钟。
注意:确保计数很少或没有,因此流式细胞仪中没有污染或残留细胞。如果有污染物,用 TBSS 冲洗更长的时间,如果仍然存在,用清洗液清洗,然后再用 TBSS。
将流速设置为 3–4 µL s -1以平均计数 300 个细胞 s -1 。必要时可以调整速度。
将细胞样品转移到流式细胞仪管中,然后加入 1 μL 的 1 mg mL -1 PI 和 1 mL 的 TBSS。快速涡旋并将样品管放入样品架中。 PI 标记允许从分析中排除死细胞,这些死细胞很容易吸收 NBD-脂质,因为它们的质膜被破坏。添加新的细胞样本后,会显示一条消息以保存刚刚收集的数据。在所使用的设备中,样品会自动以 .fcs 格式保存(查看“数据分析”以了解如何进行样品分析)。


图 2. 示意图 NBD-脂质摄取测定。
对于标记,将在 20°C 与磷脂酶抑制剂预孵育的细胞(10 6 个细胞 mL -1 )转移到含有溶解有 DMSO 的 NBD-脂质的玻璃管中,然后孵育不同的时间段。在每个时间点,取两个 100 µL 的等分试样。一份转移到预冷的 1.5-mL 空离心管中,另一份转移到预冷的含有 30 µL 20% BSA 的 1.5-mL 微量离心管中。随后通过流式细胞术分析样品。文本中描述了各个步骤的详细信息。
表 1. 用于对 NBD 脂质标记的哺乳动物细胞系 CHO-K1 进行流式细胞术分析的参数。 
每个参数采用的设置,包括增益、比例 (Log)、下限 (LL) 和上限 (UL)。 FSC,前向散射; SSC,侧向散射; FL1,NBD荧光通道; FL3,PI 荧光通道。使用的流式细胞仪配备有蓝色固态激光器(488 nm,20 mW )。 FL1 和 FL3 荧光分别用带通滤光片 IBP 527/30 和长通滤光片 RG 630 nm 记录。请注意,这些值可能需要根据所使用的细胞系和/或流式细胞仪进行调整。
参数_增益_日志_L -LU -L
FSC _1 70升在1 109 99.9
不锈钢_1 90升在1 09 99.9
F L12 20日志41 09 99.9
F L34 15日志41 07 35.9
荧光脂质代谢分析
可以根据修改后的(Bligh and Dyer, 1959)方案进行脂质提取。该方法基于在氯仿和甲醇的双相混合物中分配脂质。添加有机溶剂(如氯仿)后,甲醇会破坏脂质和蛋白质之间的氢键。除了离心,所有描述的步骤都在流动柜中进行,以避免直接暴露于有机溶剂。
在给定时间点转移等分的细胞悬浮液 至 12 mL 玻璃管。我们通常在 60 分钟后分析代谢,取与 ~4 nmol NBD-脂质相对应的体积(150 µL 用 NBD-PE 标记的细胞;300 µL 用 NBD-PC 标记的细胞;600 µL 用 NBD-PS 标记的细胞或 NBD-SM)。 
考虑到每个玻璃管中已经存在的体积,最多添加 2 mL 的 ddH 2 O。
添加 2.2 mL 甲醇,然后使用玻璃刻度移液器添加 2 mL 氯仿,并小心涡旋 3–5 s,以实现单相。
× g离心管 5 分钟以获得两个分离的相。
使用玻璃巴斯德吸管将下相(含有脂质的氯仿相)转移到锥形底部玻璃管中。
对于第二轮萃取,在原管中加入 1 mL 氯仿,短暂涡旋,再次以 500 × g离心5 分钟。
将下层相从步骤 D5 转移到玻璃管中。
准备四个锥形底玻璃,并添加 4 nmol 的 NBD-PC、NBD-PE、NBD-PS 和 NBD-SM 重新悬浮在氯仿中:甲醇(1:1,v/v)来自用于标记细胞的相同脂质库存。这些样品将作为标准品与提取的脂质样品进行比较。
将脂质提取物和标准样品在 250 mbar 真空下干燥过夜 或在温和的氮气流下 30 分钟至 1 小时。
在 TLC 硅胶板上,用铅笔将 1 厘米线标记为加载点。它们应与底边相距 1.5 厘米,与侧边相距 1 厘米且彼此相距 1 厘米。总共绘制 8 条线,其中 4 条用于 NBD 脂质标准,4 条用于提取的 NBD 脂质样品(图 3A)。样品信息可以用铅笔写在装载点下。 在 TLC 板上书写时不要用力过大,因为这会去除二氧化硅涂层。
小心地将运行缓冲液(参见食谱)倒入显影室中,直到填充到约 0.5 厘米高。盖上盖子,让空气饱和约 20 分钟。
μL的氯仿/甲醇 (1:1, v/v)的体积中。
使用玻璃巴斯德移液器将溶解的 NBD 脂质涂抹到 TLC 二氧化硅板上,小心地将其放在 1 厘米加载点的顶部,以避免剥落二氧化硅并确保均匀分布。在加样点顶部添加更多样品之前,让有机溶剂蒸发。
放置 TLC 板 尽可能均匀地在腔室中稍微倾斜。盖上盖子并运行 20-25 分钟,或直到运行前端距离顶部边缘 1 厘米。
从腔室中取出 TLC 板,用铅笔标记运行前部,让它干燥 15-30 分钟。
在 Chemidoc 成像系统中使用 Image Lab TM软件和发射滤光片 530/28 nm 在蓝光 Epi 照明下成像 NBD-脂质荧光(图 5C)。
注意:将 TLC 板放在透明塑料袋的顶部,以免刮伤 Chemidoc 托盘表面。
TM软件对条带强度进行量化,例如,以评估脂质代谢转化的百分比。低水解 (<10%) 不影响 NBD-脂质内化动力学。然而,对于更高的水解 (>10%),确定转化是发生在细胞内还是在细胞表面,以解释结果是至关重要的。


图 3. TLC 的制备。
A ) 用铅笔在二氧化硅板上标记加载点,并用玻璃巴斯德吸管加载 NBD-脂质(详细信息见正文)。车道不应放置得太靠近边缘:保持 1.5 厘米的距离,如红色箭头所示。 B ) 将硅胶板放入含有碱性电泳缓冲液的 TLC 室中,并盖上玻璃盖。将板显影直到溶剂在板顶部下方约半厘米处。
数据分析
从流式细胞仪中以原始格式 (.fcs) 导出数据并导入 FlowJo 软件。
双击第一个文件的顶部。将打开一个图,并且可以将单元格可视化为点。分别为 x 和 y 轴选择 FCS 和 SSC ,并选择对数刻度。调整比例范围。
周围生成一个门并将其标记为 P1,在这种情况下指的是 CHO-K1 细胞(图 4A)。双击所选种群的顶部,将打开另一个面板图。
为 x 轴选择 FL3(PI 荧光),并在新图中为 y 轴保留 SSC。在具有低 PI 荧光的细胞群周围生成一个门,其中仅包括活细胞,并标记为 P2(图 4B)。
双击 P2 种群的顶部。一个新的情节将打开。为 x 轴选择 FL1(NBD-脂质荧光),为 y 轴选择 SSC(侧向散射)(图 4C)。
在 FlowJo 界面中,创建的种群 P1 和 P2 出现在 .fcs 文件下方。右键单击每个文件并选择“将分析复制到组”以将门应用于所有其他文件。
检查生成的门是否适合所有样本,并在必要时移动以适合。
在 FlowJo 界面中,点击 Workplace,然后点击 Add Statistics。选择“几何平均值”、总体 P2 和参数 FL1。
将分析的样本保存为 Worspace (WSP) 文件和 Excel (xls) 文件。
计算细胞在每个时间点吸收的 NBD-脂质百分比 (图 5A, B)。没有添加 BSA 的样品的每个时间点的几何平均值被认为是 100% NBD 荧光。在给定时间点具有 BSA 的样本的几何平均值是与分配给 100% 的值相比的相对百分比(来自没有 BSA 的样本;等式 1)。新计算的百分比在步骤 11 和方程式中称为不可提取的 NBD-脂质。 1 或图 5B 和其他地方的 NBD 脂质摄取。绘制每个时间点内化 NBD 脂质的百分比(图s 5A 和 5B)。
Non˗extractable NBD˗lipid (%)=(geometrical mean value of sample with BSA)/(geometrical mean value of sample without BSA)  × 100     Eq.1
根据单指数曲线的方程拟合数据(例如, 使用 Microsoft Excel 和插件程序 Solver):
y=a+b*(1-e^(c*t) )                   Eq.2
在哪里 
y是在每个时间点以百分比 (%) 表示的 NBD-脂质荧光的拟合值,
t是添加 NBD 脂质后的时间,
a是 t = 0 时不可提取的 NBD 脂质的百分比,
b是稳态下不可提取的 NBD 脂质的百分比,
c是比率系数。


图 4.使用FlowJo软件对细胞群进行门控。
细胞用 NBD 脂质标记并在给定时间点收集。该数据说明了与标记为 60 分钟并用 BSA 处理的细胞相对应的门控程序。 A ) 细胞在侧向和前向散射的对数刻度图中可视化。群体P1 被定义为对应于哺乳动物细胞并且不包括其他可能的颗粒(例如,细胞碎片)。 B ) P1 群体在绘制侧向散射和红色碘化丙啶 (PI; dos Santos等人, 2013 )-荧光的新面板中可视化。通过选择活细胞并排除死细胞来定义群体 P2,并用 PI 标记。 C )随后将 P2 群体绘制在侧向散射和绿色 NBD 荧光板上,从而显示内化 NBD 脂质的活细胞。


图 5. CHO-K1 细胞中 NBD 脂质摄取和脂质代谢的示例性时间流逝。
在反萃取至 BSA 后,通过流式细胞术分析在 20°C 下用指定的 NBD-脂质培养的细胞。 A )在指定时间点测量 BSA 孵育后 CHO-K1 细胞中 NBD-PC 荧光强度的直方图。请注意,如果没有代谢修饰,例如,NBD-脂质类似物水解成溶血衍生物和游离 NBD-脂肪酸,未经 BSA 处理的细胞的相应直方图将显示随时间持续高的细胞相关荧光. B ) NBD-脂质在 CHO-K1 细胞中内化的时间过程,显示为荧光强度相对于在没有 BSA 的情况下处理的细胞的荧光的百分比。绘制的线代表单指数曲线的最佳拟合。 C )孵育 60 分钟和 BSA 处理后从 CHO-K1 细胞中提取的 NBD 脂质的薄层色谱图。脂质样品与标准品一起被解析到硅胶板上,并进行荧光成像。二酰基甘油 (DAG) 神经酰胺 ( Cer ) 和 NBD-X [6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid] (hexanoic acid),NBD 产品-脂质代谢转化也被指出。星号 (*) 表示来自 NBD-PE 的未知污染物或脂质种类。右边的箭头表示碱性缓冲液的运行方向。标有运行原点 (O) 和运行前沿 (F)。 NBD,硝基苯并恶二唑; PC、磷脂酰胆碱; PE、磷脂酰乙醇胺; PS,磷脂酰丝氨酸; SM,鞘磷脂。
食谱
使用内部水蒸馏系统获得的双蒸馏水 (ddH 2 O) 制备缓冲液。或者,所有缓冲液均使用超纯水制备,25°C 时纯化灵敏度为 18 MΩ cm -1 。
细胞培养基
打开 500 mL 烧瓶的高葡萄糖 DMEM 培养基
加入50 mL胎牛血清
加入 5 mL L-谷氨酰胺
加入 5 mL 青霉素-链霉素
在无菌柜中制备;储存于 4°C
TBSS 缓冲器
136 毫米氯化钠
2.6 毫米氯化钾
1.8 毫米氯化钙2
1 毫米氯化镁2
0.36 毫米 NaH 2 PO 4
5.56 毫米葡萄糖
5 毫米 HEPES
用 NaOH 将 pH 值调节至 7.4。完成体积至 1 L。使用 0.22 µm 过滤器过滤进行消毒。
在 4°C 下可保存数月。
NBD-脂质库存
所有步骤都必须在玻璃管中进行,以防止脂质的非特异性结合。
注意:氯仿是一种化学危险品。不要吸入气体/烟雾/蒸气/喷雾。穿合适的防护服。在通风橱中工作。
使用玻璃注射器将所需量的 NBD 脂质转移到直径为 12 毫米的一次性玻璃管中。通常,我们用 80 nmol 的 NBD-PE、40 nmol 的 NBD-PC 和 20 nmol 的 NBD-PS 或 NBD-SM 标记 3 mL 的细胞悬浮液。
笔记: 这些数量已经过优化,以允许在分析标记细胞时使用相同的流式细胞仪设置。 
在 250 mbar 真空下过夜或在温和的氮气流下干燥脂质30 分钟至 1 小时,以便在管底部形成干燥的脂质膜。
在使用前不久将 NBD-脂质重新悬浮在 20 μL 的 DMSO 中。将移液器尖端放在玻璃管壁和干燥的脂质膜顶部,以圆形方式添加 DMSO。缓慢上下移液器重复圆周运动,直到所有脂质膜重新悬浮。
注意: DMSO 脂质悬浮液由于其吸湿性而容易沉淀,应新鲜制备。 DMSO 是一种化学危险品。穿合适的防护服。
200 mM PMSF 库存
重量 34.838 毫克,带防护装备,避免吸入或接触皮肤。
溶解在 1 m L乙醇中。
储存在-20°C。
注意: PMSF 是一种化学危险品,特别是在其固态时,如果吞食会产生剧毒,接触会导致严重的皮肤灼伤和眼睛损伤。准备库存溶液时,穿上合适的防护服并在通风橱中工作。小心处理原液。
5 mM 的 OBAA 库存
重量 2.14 毫克
加入 1 mL 无水乙醇
储存于 -80°C
注意:穿合适的防护服。
TBSS 中的 BSA(不含香精脂肪酸,20% w/v)
重量 200 mg BSA 在 15 mL Falcon 管中
添加 1 mL 的 TBSS
在 37°C 的水浴中孵育直至部分溶解。
在冰箱中储存至第二天,并在一周内使用;不要冻结溶液。
PI 库存 1 mg mL -1
重量 1 毫克 PI
添加 1 mL 的 ddH 2 O
储存于 -20°C
注意: PI 是一种疑似致癌物,应小心处理。染料必须按照适用的当地法规安全处置。 穿合适的防护服。
碱性运行缓冲液
将以下体积混合在 30:35:35:7 v/v/v/v (mL) 氯仿:乙醇:三乙胺: ddH 2 O 在蓝盖玻璃中。
用力摇晃。在实验的同一天准备。
注:TLC 腔室尺寸(宽×深×高:235 × 116 × 220 mm) 在本协议中描述的碱性运行缓冲液是通过混合 15:17.5:17.5:3.5 v/v/v/v (mL) 氯仿:乙醇:三乙胺: ddH 2 O来制备的。 有机溶剂具有化学危险性。不要吸入气体/烟雾/蒸气/喷雾。穿合适的防护服。在通风橱中工作。
致谢
该协议改编自我们之前的工作(Pomorski等人,1996;Weingartner等人,2011;dos Santos等人,2013;Grifell-Junyent等人,2022) 。这项工作得到了灵北基金会 (R221-2016-1005 to TGP) 和德国研究基金会 (GU 1133/11-1 to TGP) 的支持。 MGJ 承认哥本哈根大学和波鸿鲁尔大学研究实习交流计划的资助。我们非常感谢 Marcus Peters 博士授予我们使用流式细胞仪的权限。
利益争夺
作者声明不存在相互竞争的利益。
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引用:Herrera, S. A., Grifell-Junyent, M. and Pomorski, T. G. (2022). NBD-lipid Uptake Assay for Mammalian Cell Lines. Bio-protocol 12(4): e4330. DOI: 10.21769/BioProtoc.4330.
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