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Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
流式细胞术和共聚焦显微镜检查定量测定细胞外双链RNA摄取和亚细胞定位   

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本实验方案简略版
Immunity
Sep 2017

Abstract

Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition receptors (PRRs) capable of detecting viral dsRNAs such as endosomal toll-like receptor 3 (TLR3) and cytosolic RIG-I-like receptors (RLRs) which lead to the production of type I interferons (IFNs). Thus, intracellular localization of viral dsRNA can provide insight into the downstream signaling pathways leading to innate immune activation. Here, we describe a quantitative method for measuring extracellular dsRNA uptake and visualizing subcellular localization of internalized dsRNA via flow cytometry and confocal microscopy respectively.

Keywords: Double-stranded RNA (双链RNA), Endosomes (内体), Lysosomes (溶酶体), Viruses (病毒), Poly(I:C) (聚(I:C)), TLR3 (TLR3), RIG-I (RIG-I), MDA-5 (MDA-5), Confocal microscopy (共聚焦显微镜), Flow cytometry (流式细胞术)

Background

Double-stranded RNAs (dsRNAs) are a common by-product of viral replication and are potent activators of antiviral immunity via the production of type I interferon (IFN) and other pro-inflammatory cytokines (Nellimarla and Mossman, 2014). Viral dsRNAs are sensed within endosomes by TLR3 (Matsumoto et al., 2003) or in the cytosol by the RIG-I-like receptors (RLRs), RIG-I and MDA-5 (Kato et al., 2006). During lytic infections, these dsRNAs can be released into the extracellular space where they bind surface receptors on neighboring cells, such as class A scavenger receptors (SR-A) and Raftlin, and are subsequently internalized via clathrin-mediated endocytosis (Itoh et al., 2008; DeWitte-Orr et al., 2010; Watanabe et al., 2011; Dansako et al., 2013).

In our previous study, we found out that the protein SID1 transmembrane family member 2 (SIDT2) localizes to late endosomes and lysosomes and that loss of SIDT2 leads to subcellular accumulation of the synthetic dsRNA analog, poly(I:C), while not affecting initial endocytosis-mediated internalization (Nguyen et al., 2017). To do so, we developed and utilized flow cytometry and confocal microscopy-based approaches to quantitatively measure poly(I:C) uptake and subcellular localization respectively in vitro. In this protocol, we describe a further refinement of these assays to allow for high-throughput assessment of internalization and subcellular localization of different dsRNAs. These methods allow for further dissection of dsRNA trafficking during viral infection and the downstream effects of these dsRNAs on innate immune signaling.

Materials and Reagents

  1. 8 well microscope slide (ibidi, catalog number: 80826 )
  2. 24 well tissue culture plate (Corning, Falcon®, catalog number: 353047 )
  3. Sterile filtered pipette tips (0.5 µl to 1,000 µl) (Corning, Axygen, catalogue numbers: TF-300-L-R-S , TF-20-L-R-S , TF-200-L-R-S and TF-1000-L-R-S )
  4. 10 cm culture dishes (Corning, Falcon®, catalog number: 353003 )
  5. 10 ml centrifuge tubes (SARSTEDT, catalog number: 62.9924.284 )
  6. 1.5 ml microcentrifuge tubes (Sigma-Aldrich, catalog number: EP0030120086 )
  7. 1.2 ml Micro Titertube (Thermo Fisher Scientific, Quality Scientific Plastics, catalog number: 845-Q )
  8. Serological pipettes, individually wrapped, 10 ml (Corning, Falcon®, catalog number: 356551  )
  9. Mammalian cell line of interest, here: mouse embryonic fibroblasts (see Note 1)
  10. 70% (v/v) ethanol (Chem Supply, catalog number: EA043 )
  11. Dulbecco’s modified Eagle medium (DMEM) or other suitable complete growth medium for culture of cell line of interest
  12. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F9423 )
  13. Phosphate-buffered saline (PBS) (sterile) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  14. Penicillin/streptomycin solution (Sigma-Aldrich, catalog number: P4333 )
  15. Poly(I:C)-fluorescein (InvivoGen, catalog number: tlrl-picf )
  16. Poly(I:C)-rhodamine (InvivoGen, catalog number: tlrl-picr )
  17. dsRNA specific monoclonal antibody (J2, SCICONS English and Scientific Consulting, catalog number: 10010200 )
  18. RNase A enzyme (Sigma-Aldrich, catalog number: R4875 )
  19. 1x trypsin-EDTA solution (Sigma-Aldrich, catalog number: 59430C )
  20. Paraformaldehyde (PFA) powder (Sigma-Aldrich, catalog number: 158127 )
  21. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  22. DAPI (4’,6-Diamidine-2’-phenylindole dihydrochloride) powder (Sigma-Aldrich, catalog number: D9542 )
  23. ImmersolTM Immersion Oil (Carl Zeiss, catalog number: 4449620000000 )
  24. Complete growth medium (see Recipes)
  25. 10% FBS/PBS (see Recipes)
  26. 4% paraformaldehyde (w/v) (see Recipes)
  27. Permeabilization buffer (see Recipes)
  28. DAPI solution (see Recipes)

Equipment

  1. Pipetting aid (Thermo Fisher Scientific, catalog number: 9531 )
  2. Micropipettes from 0.5 µl to 1 ml (Mettler-Toledo International, Rainin, model: Pipet-LiteTM XLS+ )
  3. Hemocytometer
  4. Class II biological safety cabinet/tissue culture hood
  5. Humidified CO2 incubator (95% air, 5% CO2, 37 °C)
  6. Inverted light microscope (phase contrast)
  7. 37 °C water bath
  8. Vacuum aspiration system with glass Pasteur pipettes
  9. Table top centrifuge equipped with a swing-out rotor for 10 ml conical tubes
  10. Microcentrifuge
  11. LSRFortessa X20 (BD, BD Biosciences, model: LSRFortessaTM X-20 ) or equivalent flow cytometer
  12. LSM 780 confocal laser scanning microscope (ZEISS, model: LSM 780 ) or equivalent microscope

Software

  1. FIJI/ImageJ
  2. Zeiss ZEN package
  3. Microsoft Excel
  4. FlowJo
  5. GraphPad Prism 7

Procedure

  1. Cell culture and maintenance
    1. Perform all cell culture-based work in a class II biological safety cabinet/tissue culture hood. Ensure work surface and materials are sterilized using 70% (v/v) ethanol.
    2. Grow mammalian cells of choice in complete growth medium using 10 cm cell culture dishes or cell culture flasks in a humidified CO2 incubator (95% air, 5% CO2, 37 °C). Here, we use mouse embryonic fibroblasts (MEFs) derived from C57BL/6 mice and MEFs lacking the dsRNA transporter, SIDT2, as our gene of interest (see Note 2).
    3. Maintain cells using standard cell culture procedures or as recommended by the supplier. Here, split MEFs approximately every 3-4 days or before cells reach 100% confluency using standard cell culture procedures (see Note 3).
    4. One day prior to stimulation, split the cells as above and determine cell number using a hemocytometer or other appropriate methods under a light microscope.

  2. Quantification of poly(I:C) uptake by flow cytometry
    1. Seed 1 x 104 cells in a 24 well plate in triplicate for the following conditions in a total volume of 500 µl of complete growth medium (cells may be seeded for additional conditions as required):
      1. Unstimulated
      2. Stimulated with poly(I:C) (see Note 4)
    2. Let cells adhere and rest overnight in a humidified CO2 incubator (95% air, 5% CO2, 37 °C).
    3. The next morning, remove cells from the incubator and add 1 µg/ml of fluorescein-poly(I:C) to the cell culture medium (see Note 5).
    4. Stimulate cells in an incubator at 37 °C, 5% CO2 for 24 h.
    5. After 24 h of stimulation, carefully aspirate complete growth medium from each well with a glass Pasteur pipette using a vacuum aspiration system.
    6. Wash cells with 1 ml of cold 1x PBS using a P1000 pipette. Carefully add PBS on the side of the well to avoid dislodging cells.
    7. Repeat Step B6 for a total of 3 washes.
    8. Harvest cells by adding 500 µl of Trypsin to each well and incubate cells at 37 °C, 5% CO2 for 5 min.
    9. Add 500 µl of complete growth medium to each well.
    10. Gently resuspend cells by pipetting up and down 3-5 times.
    11. Transfer resuspended cells to new individual 1.5 ml microcentrifuge tubes.
    12. Centrifuge cells in a microcentrifuge for 3 min at 500 x g and resuspend the pellet in 500 µl PBS supplemented with 10% FBS.
    13. Add 10 µl of RNase A enzyme (5 mg/ml stock) to each well at a final concentration of 100 µg/ml (see Note 6).
    14. Incubate cells at 37 °C and 5% CO2 for 30 min.
    15. Centrifuge cells in a microcentrifuge for 3 min at 500 x g.
    16. Carefully aspirate RNase A solution from each well.
    17. Resuspend cells in 1 ml cold PBS.
    18. Repeat Step B15 for a total of 3-5 washes.
    19. Resuspend cells in 200 µl of 10% FBS/PBS.
    20. Transfer cells to individual 1.2 ml Micro Titertubes and leave on ice.
    21. Analyze the incorporated fluorescence of cells of both genotypes and using flow cytometry. Compare the histograms and corresponding mean fluorescence intensities (MFI) between fluorescein-poly(I:C)-stimulated cells and unstimulated cells (Figure 1).


      Figure 1. Analysis of poly(I:C)-fluorescein uptake by flow cytometry. The endocytic activity of WT and Sidt2-/- MEFs was assessed by measuring the uptake of fluorescein-conjugated poly(I:C) following 24 h stimulation. A. Representative histograms for the mean fluorescence intensities (MFI) for each genotype are shown. B. Mean percentage of poly(I:C)-fluorescein positive cells. n = 3 technical replicates and errors bars represent ± SEM. Loss of SIDT2 did not affect poly(I:C) uptake.

  3. Confocal analysis of poly(I:C) subcellular localization
    1. Seed 5 x 103 cells in an 8 well chamber slide for the following conditions in a total volume of 200 µl of complete growth medium (cells may be seeded for additional conditions as required):
      1. Unstimulated
      2. Stimulated with poly(I:C)
    2. The next morning, remove cells from the incubator and add 1 µg/ml of rhodamine-poly(I:C) to the cell culture medium (see Note 5).
    3. Incubate cells at 37 °C, 5% CO2 for 24 h.
    4. Carefully aspirate and discard complete growth medium from each well with a P200 pipette.
    5. Wash cells with 200 ml of cold 1x PBS using a P1000 pipette.
    6. Repeat Step C5 for a total of 3 washes.
    7. After washes, add 200 µl PBS supplemented with 10% FBS to each well.
    8. Add 10 µl of RNase A enzyme (5 mg/ml stock) to each well at a final concentration of 100 µg/ml (see Note 6).
    9. Incubate cells at 37 °C and 5% CO2 for 30 min.
    10. Carefully aspirate RNase A solution from each well.
    11. Wash cells with 200 µl cold PBS.
    12. Repeat Step C11 for a total of 3 washes.
    13. Fix cells with 200 µl of 4% PFA for 10 min on ice.
    14. Wash cells with 200 µl cold PBS.
    15. Repeat Step C14 for a total of 3 washes.
    16. Stain cells with 100 ng/ml DAPI solution in a final volume of 200 µl per well for 10 min at room temperature (see Note 7).
    17. Wash cells with 200 µl cold PBS.
    18. Repeat Step C17 for a total of 3 washes.
    19. After the final wash, add 200 µl of PBS to each well.
    20. Slides are now ready for imaging or can be stored at 4 °C for up to 2 weeks.
    21. Acquire images using a ZEISS LSM 780 confocal microscope (an equivalent microscope can be used) with a 25x oil immersion objective lens.
    22. Acquire Z-stack of 0.25 μm slices from the top to the bottom of the cells (Figure 2A).
    23. Save images as .lsm files and separate each condition into individual folders (e.g., WT untreated, WT pIC treated, KO untreated, KO pIC treated).
    24. Drag and drop all folders into a single folder (see Note 8).
    25. Images are now ready for analysis using FIJI/ImageJ software (see below).

Data analysis

  1. Determine appropriate threshold value for images
    1. Open an image file that contains cells with representative poly(I:C)-rhodamine or alternative dsRNA signal intensity in FIJI/ImageJ.
    2. Acquire maximum projection image (Navigate to toolbar on the top left corner of the screen → Image → Stacks → Z Project → Projection type: Max intensity → Okay).
    3. Select channel corresponding to poly(I:C)-rhodamine by adjusting the C slider on the bottom of the maximum projection image window.
    4. From the toolbar, select Image → Adjust → Threshold…
    5. Set bottom slider to 255.
    6. Adjust top slider until threshold (red) is able to distinguish between each individual punctate endosome (Figure 2B). Here, we used a threshold of 60.
    7. Note down threshold value.


      Figure 2. Analysis of poly(I:C)-rhodamine subcellular localization by confocal microscopy. A. WT and Sidt2-/- MEFs were treated with rhodamine-conjugated poly(I:C) for 24 h and assessed via confocal microscopy. Representative maximum projection images are shown, red = poly(I:C), blue = DAPI. B. Snapshot from FIJI/ImageJ showing selection of threshold value step.

  2. Subcellular localization quantification
    1. Download FIJI macro file from: https://bitbucket.org/DrLachie/rna_subcell.
    2. Open macro file in FIJI/ImageJ.
    3. Change ‘GreenThreshold’ to the threshold value determined in the above section (e.g., ‘60’).
    4. Click ‘Run’.
    5. Open output\ folder and check the accuracy of cell segmentation (Figure 3A) and individual cell by cell quantification of the punctate area, intensity and number of puncta determined by macro.
    6. Open results.xls file located in top folder using Microsoft Excel.
    7. Calculate and Graph results for the average punctate area, intensity and number of puncta in Prism GraphPad or equivalent software (Figure 3B) (see Note 9).


      Figure 3. Quantification of poly(I:C)-rhodamine subcellular localization using FIJI/ImageJ macro. A. Representative output image following macro quantification showing segmentation of cells using DAPI to delineate between individual cells. Note that poly(I:C) localizes in the cytoplasm and not in the nucleus. B. Comparison of the average percentage punctate area, average intensity and average number of puncta between WT and Sidt2-/- MEFs. Data are plotted as mean ± SEM and at least 5 representative fields of view each containing 5-22 cells were analyzed per condition. **P < 0.01, ***P < 0.001.

Notes

  1. Other adherent cell lines of interest can also be used such as bone marrow-derived macrophages, HEK293T and NIH3T3, etc. We have also used non-adherent cells such as DC2.4 and bone marrow-derived dendritic cells by treating suspension cells in 1.5 ml microcentrifuge tubes and subsequently mounting fixed cells onto a microscope slide via a cytospin. However, we find that this method leads to a loss of cell morphology and therefore recommend the use of adherent cells if possible.
  2. We have previously reported that the loss of SIDT2 leads to accumulation of dsRNA within endosomes (Nguyen et al., 2017). Here, Sidt2-/- MEFs were used to assess endosomal subcellular localization compared to WT MEFs. Cells lacking or overexpressing gene of interest can be used in place of Sidt2-/- MEFs as desired.
  3. Here, we pre-warm 1x PBS, 1x Trypsin solution and complete growth medium to 37 °C using a water bath. Old medium is removed from the cell culture dish using a Pasteur pipette by vacuum aspiration. Cells are subsequently washed with 10 ml of 1x PBS before the addition of 3 ml (per 10 cm dish) of Trypsin solution and incubate for 3-5 min at 37 °C. Detached cells are resuspended in 10 ml/10 cm dish by pipetting 2-3 times up and down and transfer to a 10 ml centrifuge tube. Cells are pelleted in a tabletop centrifuge at 500 x g, 5 min at RT and resuspended in 10 ml of fresh complete growth medium. For cell maintenance, add 1 ml of cell suspension to a new 10 cm cell culture dish with 10 ml of complete growth medium and incubate in a humidified CO2 incubator (95% air, 5% CO2, 37 °C).
  4. Here, rhodamine or fluorescein conjugated poly(I:C) (InvivoGen) was used to stimulate cells as it is commercially available and readily accessible. Alternate fluorescently-tagged ligands can also be used as required. We have also successfully detected viral dsRNA via immunofluorescence staining using a dsRNA specific monoclonal antibody (J2, English and Scientific Consulting).
  5. The concentration of poly(I:C) and length of stimulation should be optimized according to cell line of interest. We found that MEFs do not efficiently internalize poly(I:C) and therefore require a 24 h stimulation time. However, bone marrow-derived dendritic cells and macrophages require much shorter stimulation times (1 to 3 h) for sufficient uptake.
  6. Treating cells with RNase will degrade any surface-bound dsRNA while retaining internalized dsRNA. Here, we used RNase A which is able to cleave both ssRNA and dsRNA at low salt concentrations. Alternatively, RNase III can be used to specifically cleave dsRNA.
  7. It is important to stain cells with DAPI in the presence of detergent to permeabilize the cell membrane and allow DAPI access to nuclear DNA. We use 0.1% Tween to permeabilize cells; however, alternative detergents can be used such as Saponin or Triton-X. 

  8. The FIJI macro used for image analysis requires two layers of folders in order to proceed with analysis.
  9. Here, we demonstrate that loss of SIDT2 results in endosomal accumulation of poly(I:C), consistent with our previous findings (Nguyen et al., 2017). In that study, we also performed transient transfection and immunofluorescence staining of various endosomal markers – EEA-1 (early endosomes), RAB-7 (late endosomes) and LAMP-1 (lysosomes) – in order to precisely determine the subcellular localization of poly(I:C) within Sidt2-/- cells.

Recipes

  1. Complete growth medium
    Dulbecco’s modified Eagle medium supplemented with:
    10% fetal bovine serum
    100 U/ml penicillin
    100 µg/ml streptomycin
    Filter sterilize, store at 4 °C
  2. 10% FBS/PBS
    450 ml Sterile Phosphate Buffered Saline
    50 ml fetal bovine serum
    Store at 4 °C
  3. 4% paraformaldehyde (w/v)
    1. Dissolve 4 g of PFA powder in 90 ml PBS and heat to 65 °C while stirring. If PFA does not dissolve, add drops of 1 M NaOH until the solution becomes clear.
    2. Bring to 100 ml with PBS. Cool and filter. Aliquot and store at -20 °C. Thaw aliquots as needed and use immediately.
  4. Permeabilization buffer
    10 ml PBS
    10 µl of Tween 20
  5. DAPI solution
    1. Dissolve stock solution in sterile dH2O at a final concentration of 1 mg/ml
    2. Dilute stock solution to a final concentration of 1 µg/ml in permeabilization buffer. Use immediately

Acknowledgments

We thank the members of the Wicks and Masters labs, WEHI for helpful discussions. This protocol was adapted from Nguyen et al. (2017) Immunity 47(3):498-509.e6. DOI: 10.1016/j.immuni.2017.08.007. This work was supported by Australian NHMRC (ID 520574 and 1064591), Royal Australasian College of Physicians, Menzies Foundation, CASS Foundation (SM13-4846 and SM14- 5566), and Reid Family Trust. The authors declare no conflict of interest.

References

  1. Dansako, H., Yamane, D., Welsch, C., McGivern, D. R., Hu, F., Kato, N. and Lemon, S. M. (2013). Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells. PLoS Pathog 9(5): e1003345.
  2. DeWitte-Orr, S. J., Collins, S. E., Bauer, C. M. T., Bowdish, D. M., Mossman, K. L. (2010). An accessory to the ‘Trinity’: SR-As are essential pathogen sensors of extracellular dsRNA, mediating entry and leading to subsequent type I IFN responses. PLoS Pathog 6: e1000829.
  3. Itoh, K., Watanabe, A., Funami, K., Seya, T. and Matsumoto, M. (2008). The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-β production. J Immunol 181(8): 5522-5529.
  4. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K. J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C. S., Reis e Sousa, C., Matsuura, Y., Fujita, T. and Akira, S. (2006). Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441(7089): 101-105.
  5. Matsumoto, M., Funami, K., Tanabe, M., Oshiumi, H., Shingai, M., Seto, Y., Yamamoto, A. and Seya, T. (2003). Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 171(6): 3154-3162.
  6. Nellimarla, S. and Mossman, K. L. (2014). Extracellular dsRNA: its function and mechanism of cellular uptake. J Interferon Cytokine Res 34(6): 419-426.
  7. Nguyen, T. A., Smith, B. R. C., Tate, M. D., Belz, G. T., Barrios, M. H., Elgass, K. D., Weisman, A. S., Baker, P. J., Preston, S. P., Whitehead, L., Garnham, A., Lundie, R. J., Smyth, G. K., Pellegrini, M., O'Keeffe, M., Wicks, I. P., Masters, S. L., Hunter, C. P. and Pang, K. C. (2017). SIDT2 transports extracellular dsRNA into the cytoplasm for innate immune recognition. Immunity 47(3): 498-509 e496.
  8. Watanabe, A., Tatematsu, M., Saeki, K., Shibata, S., Shime, H., Yoshimura, A., Obuse, C., Seya, T. and Matsumoto, M. (2011). Raftlin is involved in the nucleocapture complex to induce poly(I:C)-mediated TLR3 activation. J Biol Chem 286(12): 10702-10711.

简介

双链RNA是一种有效的病原体相关分子模式(PAMP),作为病毒复制的副产物和病毒感染的众所周知的标志产生。 病毒dsRNA可以从感染的细胞释放到细胞外空间并通过胞吞作用被邻近细胞内化。 哺乳动物具有能够检测导致产生I型干扰素(IFN)的病毒dsRNA(例如内体Toll样受体3(TLR3)和胞质RIG-1样受体(RLR))的多模式识别受体(PRR)。 因此,病毒dsRNA的细胞内定位可以提供对导致先天免疫激活的下游信号传导途径的了解。 在这里,我们描述了一种测量细胞外dsRNA摄取和分别通过流式细胞仪和共聚焦显微镜观察内化dsRNA的亚细胞定位的定量方法。

【背景】双链RNA(dsRNA)是病毒复制的常见副产物,通过产生I型干扰素(IFN)和其他促炎细胞因子(Nellimarla和Mossman,2014)是抗病毒免疫的有效激活剂。病毒的dsRNA通过TLR3核内体内所感测(松本等人,2003年)或在由RIG-I样受体(RLRS),RIG-I和MDA-5(加藤等人,2006)。在裂解感染,这些dsRNA可以被释放到细胞外空间,它们结合于相邻小区,如A类清道夫受体(SR-A)和RAFTLIN表面受体,并且随后经由网格蛋白介导的内吞作用内在化(伊藤制 2008; DeWitte-Orr等人,2010; Watanabe等人,2011; Dansako等人,, >,2013)。

在我们以前的研究中,我们发现蛋白SID1跨膜家族成员2(SIDT2)定位于晚期内涵体和溶酶体,并且SIDT2的缺失导致合成的dsRNA类似物poly(I:C)的亚细胞积累,而不影响最初的内吞作用介导的内化(Nguyen等人,2017)。为此,我们开发并利用流式细胞术和基于共聚焦显微镜的方法来分别定量测量聚(I:C)摄取和亚细胞定位体外。在该协议中,我们描述了这些测定的进一步改进,以允许高通量评估不同dsRNA的内化和亚细胞定位。这些方法允许进一步解剖病毒感染期间的dsRNA运输和这些dsRNA对先天免疫信号的下游影响。

关键字:双链RNA, 内体, 溶酶体, 病毒, 聚(I:C), TLR3, RIG-I, MDA-5, 共聚焦显微镜, 流式细胞术

材料和试剂

  1. 8张显微镜载玻片(ibidi,目录号:80826)
  2. 24孔组织培养板(Corning,Falcon ,目录号:353047)
  3. 无菌过滤吸头(0.5μl-1,000μl)(Corning,Axygen,目录号:TF-300-L-R-S,TF-20-L-R-S,TF-200-L-R-S和TF-1000-L-R-S)
  4. 10厘米培养皿(康宁公司,Falcon ,目录号:353003)

  5. 10 ml离心管(SARSTEDT,目录号:62.9924.284)
  6. 1.5 ml微量离心管(Sigma-Aldrich,目录号:EP0030120086)
  7. 1.2 ml Micro Titertube(Thermo Fisher Scientific,Quality Scientific Plastics,目录号:845-Q)
  8. 血清移液管,单独包装,10毫升(Corning,Falcon <®>,产品目录号:356551 &nbsp; )
  9. 感兴趣的哺乳动物细胞系,在这里:小鼠胚胎成纤维细胞(见注1)
  10. 70%(v / v)乙醇(Chem Supply,目录号:EA043)
  11. Dulbecco改良Eagle培养基(DMEM)或其他合适的完整生长培养基用于培养感兴趣的细胞系
  12. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F9423)
  13. 磷酸盐缓冲盐水(PBS)(无菌)(Thermo Fisher Scientific,Gibco TM,目录号:14190250)
  14. 青霉素/链霉素溶液(Sigma-Aldrich,目录号:P4333)
  15. 聚(I:C) - 荧光素(InvivoGen,目录号:tlrl-picf)
  16. 聚(I:C) - 罗丹明(InvivoGen,目录号:tlrl-picr)
  17. dsRNA特异性单克隆抗体(J2,SCICONS英语和科学咨询,目录号:10010200)
  18. RNA酶A(Sigma-Aldrich,目录号:R4875)
  19. 1x胰蛋白酶-EDTA溶液(Sigma-Aldrich,目录号:59430C)
  20. 多聚甲醛(PFA)粉末(Sigma-Aldrich,目录号:158127)
  21. 吐温20(Sigma-Aldrich,目录号:P1379)
  22. DAPI(4',6-二脒-2'-苯基吲哚二盐酸盐)粉末(Sigma-Aldrich,目录号:D9542)
  23. Immersol TM浸渍油(Carl Zeiss,目录号:4449620000000)
  24. 完整的生长培养基(见食谱)
  25. 10%FBS / PBS(见食谱)
  26. 4%多聚甲醛(w / v)(见食谱)
  27. 渗透缓冲液(见食谱)
  28. DAPI解决方案(请参阅食谱)

设备

  1. 移液助剂(Thermo Fisher Scientific,目录号:9531)
  2. 0.5微升至1毫升微量移液管(梅特勒 - 托利多国际公司,Rainin,型号:Pipet-Lite TM XLS +)
  3. 血细胞计数器
  4. 二类生物安全柜/组织培养罩
  5. 加湿的CO 2培养箱(95%空气,5%CO 2,37℃)。
  6. 倒置光学显微镜(相衬)

  7. 37°C水浴
  8. 带玻璃巴斯德吸管的真空抽吸系统
  9. 台式离心机配有摆动式转子,用于10 ml锥形管
  10. 微量离心机
  11. LSRFortessa X20(BD,BD Biosciences,型号:LSRFortessa TM X-20)或等效的流式细胞仪
  12. LSM 780共聚焦激光扫描显微镜(ZEISS,型号:LSM 780)或等效显微镜

软件

  1. FIJI / ImageJ
  2. 蔡司ZEN包
  3. Microsoft Excel
  4. FlowJo
  5. GraphPad Prism 7

程序

  1. 细胞培养和维护
    1. 在第二类生物安全柜/组织培养罩中进行所有细胞培养工作。
      使用70%(v / v)乙醇确保工作表面和材料消毒。
    2. 使用10cm细胞培养皿或细胞培养瓶,在湿润的CO 2培养箱(95%空气,5%CO 2,37℃)中在完全生长培养基中培养选择的哺乳动物细胞C)。在这里,我们使用源自C57BL / 6小鼠的小鼠胚胎成纤维细胞(MEF)和缺乏dsRNA转运蛋白SIDT2的MEF作为我们感兴趣的基因(参见注释2)。
    3. 使用标准细胞培养程序或供应商推荐的细胞维持细胞。在这里,大约每3-4天或在使用标准细胞培养程序使细胞达到100%融合前分裂MEFs(见注3)。
    4. 刺激前一天,如上分裂细胞,并在光学显微镜下使用血细胞计数器或其他适当的方法测定细胞数量。

  2. 通过流式细胞术定量聚(I:C)摄取
    1. 在总体积为500μl的完全生长培养基(根据需要可以接种细胞用于附加条件)下,在24孔板中一式三份地种植1×10 4个细胞用于以下条件:
      1. 未受刺激
      2. 用聚(I:C)刺激(见注4)
    2. 让细胞粘附并在潮湿的CO 2培养箱(95%空气,5%CO 2,37℃)中静置过夜。
    3. 第二天早上,从培养箱中取出细胞并向细胞培养基中加入1μg/ ml荧光素 - 聚(I:C)(参见注释5)。
    4. 在37℃,5%CO 2下培养箱中培养24小时。
    5. 刺激24小时后,使用真空抽吸系统,用玻璃巴斯德吸管小心地从每个孔中吸出完整的生长培养基。
    6. 使用P1000移液管用1ml冷的1x PBS清洗细胞。
      小心地在孔的一侧添加PBS以避免细胞脱落。
    7. 重复步骤B6共3次。
    8. 通过向每个孔中加入500μl胰蛋白酶收获细胞,并在37℃,5%CO 2下孵育细胞5分钟。

    9. 每孔加入500μl完全生长培养基
    10. 通过上下移动3-5次轻轻地重悬细胞。
    11. 将重新悬浮的细胞转移到新的1.5 ml微量离心管中。
    12. 在500×g的微量离心机中离心细胞3分钟,并将沉淀重悬于500μl补充有10%FBS的PBS中。
    13. 每孔加入10μlRNA酶A(5mg / ml储存液),终浓度为100μg/ ml(见注6)。
    14. 在37℃和5%CO 2下培养细胞30分钟。
    15. 在500×g的微量离心机中离心细胞3分钟。
    16. 小心地吸取每口井的RNase A溶液。
    17. 将细胞重悬于1 ml冷PBS中。
    18. 重复步骤B15,共洗涤3-5次。
    19. 将细胞重悬于200μl10%FBS / PBS中。
    20. 将细胞转移到单独的1.2毫升Micro Titertube中,并放置在冰上。
    21. 分析两种基因型细胞的掺入荧光并使用流式细胞术。比较荧光素 - 聚(I:C) - 刺激的细胞和未刺激的细胞(图1)之间的直方图和相应的平均荧光强度(MFI)。


      图1.通过流式细胞术分析聚(I:C) - 荧光素摄取 WT和 Sidt2 MEF的内吞活性通过在24小时刺激后测量荧光素 - 缀合的聚(I:C)的摄取来评估。 A.显示每个基因型的平均荧光强度(MFI)的代表性直方图。 B.聚(I:C) - 荧光素阳性细胞的平均百分比。 n = 3的技术重复和错误条代表±SEM。 SIDT2的丢失不影响聚(I:C)摄取。

  3. 聚(I:C)亚细胞定位的共聚焦分析
    1. 在总体积为200μl的完全生长培养基(根据需要可以接种细胞用于附加条件)下,在8孔室中将5×10 3个细胞种植于以下条件下:
      1. 未受刺激
      2. 用聚(I:C)刺激
    2. 第二天早上,从培养箱中取出细胞,并将1μg/ ml罗丹明 - 聚(I:C)加入到细胞培养基中(见注5)。

    3. 在37°C,5%CO 2下孵育细胞24小时。

    4. 用P200移液器小心吸出并丢弃每个孔中的完整生长培养基

    5. 使用P1000移液器用200毫升冷的1x PBS清洗细胞。
    6. 重复步骤C5,共3次洗涤。
    7. 洗涤后,每孔加入200μl补充有10%FBS的PBS。
    8. 每孔加入10μlRNA酶A(5mg / ml储存液),终浓度为100μg/ ml(见注6)。
    9. 在37℃和5%CO 2下培养细胞30分钟。
    10. 小心地吸取每口井的RNase A溶液。
    11. 用200μl冷PBS洗细胞。
    12. 重复步骤C11共3次。

    13. 用200μl4%PFA在冰上固定细胞10分钟。
    14. 用200μl冷PBS洗细胞。
    15. 重复步骤C14共3次。
    16. 用100 ng / ml DAPI溶液染色细胞,最终体积为200μl/孔,室温下10 min(见注7)。
    17. 用200μl冷PBS洗细胞。
    18. 重复步骤C17共3次。
    19. 最后一次洗涤后,每孔加入200μlPBS。
    20. 幻灯片现在可以进行成像,或者可以在4°C下保存2周。
    21. 使用ZEISS LSM 780共聚焦显微镜(可使用等效显微镜)和25倍油浸物镜采集图像。

    22. 从细胞的顶部到底部收集0.25μm切片的Z-Stack(图2A)。
    23. 将图像保存为.lsm文件,并将每个条件分隔为单独的文件夹(例如,WT未处理,WT pIC处理,未处理,KO pIC处理)。
    24. 拖放所有文件夹到一个文件夹中(见注8)。
    25. 现在图像可以使用FIJI / ImageJ软件进行分析了(见下文)。

数据分析

  1. 确定图像的适当阈值
    1. 在FIJI / ImageJ中打开包含具有代表性聚(I:C) - 罗丹明或替代dsRNA信号强度的细胞的图像文件。
    2. 获取最大投影图像(导航到屏幕左上角的工具栏→图像→堆栈→Z项目→投影类型:最大强度→好)。
    3. 通过调整最大投影图像窗口底部的C滑块,选择与poly(I:C) - 罗丹明对应的通道。
    4. 从工具栏中选择图像→调整→阈值...
    5. 将底部滑块设置为255.
    6. 调整顶部滑块,直到阈值(红色)能够区分每个单独的点状内体(图2B)。在这里,我们使用了一个60的门槛。
    7. 记下临界值。


      图2.通过共聚焦显微镜分析聚(I:C) - 罗丹明亚细胞定位A.WT和 Sidt2 - / -

  2. 亚细胞定位定量
    1. https://bitbucket.org/DrLachie/rna_subcell 下载FIJI宏文件。
    2. 在FIJI / ImageJ中打开宏文件。
    3. 将'GreenThreshold'更改为上面确定的阈值(,例如,'60')。
    4. 点击“运行”。
    5. 打开输出\文件夹并检查细胞分割的准确性(图3A)和单个细胞逐点定量点的面积,强度和由宏确定的点数。
    6. 使用Microsoft Excel打开位于顶层文件夹中的results.xls文件。
    7. 在Prism GraphPad或同等软件(图3B)中计算并绘制平均点状面积,强度和点数的结果(见注9)。


      图3.使用FIJI / ImageJ宏定量聚(I:C) - 罗丹明亚细胞定位A.宏观定量后的代表性输出图像,显示使用DAPI描绘单个细胞之间的细胞分割。请注意poly(I:C)定位于细胞质而不是细胞核。 B.比较野生型和野生型之间平均斑点面积百分比,平均强度和平均斑点数目。数据绘制为平均值±SEM,并且在每个条件下分析至少5个代表性视野,每个包含5-22个细胞。 ** P &lt; 0.01,*** 0.001。

笔记

  1. 也可以使用其他感兴趣的贴壁细胞系,例如骨髓来源的巨噬细胞,HEK293T和NIH3T3等等。通过在1.5ml微量离心管中处理悬浮细胞并随后通过细胞离心涂片将固定细胞安装到显微镜载玻片上,我们还使用非贴壁细胞如DC2.4和骨髓衍生的树突细胞。但是,我们发现这种方法会导致细胞形态的损失,因此建议尽可能使用贴壁细胞。
  2. 我们以前曾报道SIDT2的丢失导致dsRNA在核内体内积聚(Nguyen等人,2017)。在这里,与WT MEF相比,使用MEFs评估内体亚细胞定位。缺乏或过表达感兴趣基因的细胞可以根据需要用于代替 - / - MEFs。
  3. 在这里,我们使用水浴预热1x PBS,1x胰蛋白酶溶液并将生长培养基完全培养至37℃。使用巴斯德移液管通过真空抽吸将旧培养基从细胞培养皿中移出。随后用10ml 1x PBS洗涤细胞,然后加入3ml(每10cm培养皿)的胰蛋白酶溶液并在37℃孵育3-5分钟。将分离的细胞重悬于10ml / 10cm培养皿中,上下移液2-3次并转移至10ml离心管中。将细胞在台式离心机中500×g,5分钟在室温下沉淀并重新悬浮于10ml新鲜完全生长培养基中。为了细胞维持,将1ml细胞悬液加入到具有10ml完全生长培养基的新的10cm细胞培养皿中,并在湿润的CO 2培养箱(95%空气,5%CO > 2℃,37℃)。
  4. 在这里,罗丹明或荧光素共轭聚(I:C)(InvivoGen)用于刺激细胞,因为它是可商购的并且容易获得。根据需要也可以使用替代荧光标记的配体。我们还使用dsRNA特异性单克隆抗体(J2,英语和科学咨询)通过免疫荧光染色成功检测到病毒dsRNA。
  5. 聚(I:C)的浓度和刺激的长度应根据感兴趣的细胞系优化。我们发现MEFs不能有效地内化poly(I:C),因此需要24小时的刺激时间。然而,骨髓来源的树突状细胞和巨噬细胞需要更短的刺激时间(1到3小时)以获得足够的摄取。
  6. 用RNA酶处理细胞会降解任何表面结合的dsRNA,同时保留内化的dsRNA。在这里,我们使用能够在低盐浓度下裂解ssRNA和dsRNA的RNA酶A.或者,RNA酶III可用于特异性切割dsRNA。
  7. 在洗涤剂的存在下用DAPI染色细胞以使细胞膜透化并允许DAPI接近核DNA是很重要的。我们使用0.1%Tween来透化细胞;然而,可以使用替代的洗涤剂,例如皂苷或Triton-X。
  8. 用于图像分析的FIJI宏需要两层文件夹才能继续进行分析。
  9. 在这里,我们证明SIDT2的丢失导致poly(I:C)的内体积累,这与我们先前的发现(Nguyen et al。 2017)一致。在该研究中,我们还进行了各种内体标记--EEA-1(早期内体),RAB-7(晚期内体)和LAMP-1(溶酶体)的瞬时转染和免疫荧光染色 - 以精确确定聚(I:C)在 Sidt2 - / - 单元格内。

食谱

  1. 完整的生长培养基
    Dulbecco的改良Eagle培养基补充:
    10%胎牛血清
    100U / ml青霉素
    100μg/ ml链霉素
    过滤消毒,在4°C储存
  2. 10%FBS / PBS
    450毫升无菌磷酸盐缓冲盐水
    50毫升胎牛血清
    在4°C储存
  3. 4%多聚甲醛(w / v)
    1. 将4克PFA粉末溶于90毫升PBS中并在搅拌下加热至65℃。如果PFA不溶解,加入1 M NaOH溶液直至溶液变澄清。
    2. 带上100毫升PBS。冷却并过滤。分装并储存在-20°C。根据需要解冻等分试样并立即使用。
  4. 透化缓冲液
    10毫升PBS
    10μl吐温20
  5. DAPI解决方案
    1. 以1 mg / ml的终浓度将原液溶解在无菌dH 2 O中。

    2. 在透化缓冲液中稀释原液至终浓度为1μg/ ml。立即使用

致谢

我们感谢Wicks和Masters实验室的成员WEHI进行了有益的讨论。该协议改编自Nguyen等人(2017)免疫 47(3):498-509.e6。 DOI:10.1016 / j.immuni.2017.08.007。这项工作得到了澳大利亚NHMRC(ID 520574和1064591),澳大利亚皇家内科学院,孟席斯基金会,中国社会科学院基金会(SM13-4846和SM14- 5566)和瑞德家族信托基金会的支持。作者宣称没有利益冲突。

参考

  1. Dansako,H.,Yamane,D.,Welsch,C.,McGivern,D.R.,Hu,F.,Kato,N.and Lemon,S.M。(2013)。 A类清道夫受体1(MSR1)通过介导toll样受体3识别来限制丙型肝炎病毒的复制在相邻细胞中产生的病毒RNAs。 PLoS Pathog 9(5):e1003345。
  2. DeWitte-Orr,S.J.,Collins,S.E.,Bauer,C.M.T.,Bowdish,D.M。,Mossman,K.L。(2010)。 “Trinity”的附件:SR-As是细胞外dsRNA的重要病原体传感器,能够介导进入并导致随后的I型IFN反应。
  3. Itoh,K.,Watanabe,A.,Funami,K.,Seya,T。和Matsumoto,M。(2008)。 网格蛋白介导的内吞途径参与dsRNA诱导的IFN-β产生。 < J Immunol 181(8):5522-5529。
  4. Kato,H.,Takeuchi,O.,Sato,S.,Yoneyama,M.,Yamamoto,M.,Matsui,K.,Uematsu,S.,Jung,A.,Kawai,T.,Ishii,KJ,Yamaguchi ,O.,Otsu,K.,Tsujimura,T.,Koh,CS,Reis e Sousa,C.,Matsuura,Y.,Fujita,T。和Akira,S。(2006)。 MDA5和RIG-I解旋酶在识别RNA病毒中的差别作用 Nature 441(7089):101-105。
  5. Matsumoto,M.,Funami,K.,Tanabe,M.,Oshiumi,H.,Shingai,M.,Seto,Y.,Yamamoto,A。和Seya,T。(2003)。 Toll样受体3在人树突状细胞中的亚细胞定位 J免疫学 171(6):3154-3162。
  6. Nellimarla,S。和Mossman,K.L。(2014)。 细胞外dsRNA:细胞摄取的功能和机制 J干扰素细胞因子Res 34(6):419-426。
  7. Nguyen,TA,Smith,BRC,Tate,MD,Belz,GT,Barrios,MH,Elgass,KD,Weisman,AS,Baker,PJ,Preston,SP,Whitehead,L.,Garnham,A.,Lundie,RJ, Smyth,GK,Pellegrini,M.,O'Keeffe,M.,Wicks,IP,Masters,SL,Hunter,CP和Pang,KC(2017)。 SIDT2将胞外dsRNA转运到细胞质中进行先天性免疫识别。 免疫< 47(3):498-509 e496。
  8. Watanabe,A.,Tatematsu,M.,Saeki,K.,Shibata,S.,Shime,H.,Yoshimura,A.,Obuse,C.,Seya,T。和Matsumoto,M.(2011)。 Raftlin参与核捕获复合物诱导poly(I:C)介导的TLR3激活。 / J> J Biol Chem 286(12):10702-10711。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nguyen, T. A., Whitehead, L. and Pang, K. C. (2018). Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy. Bio-protocol 8(12): e2890. DOI: 10.21769/BioProtoc.2890.
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