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Jan 2018

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Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
流式细胞术测定TLR4和CD14受体内吞作用   

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Abstract

After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.

Keywords: Toll-like receptor (Toll-类受体), TLR (TLR), TLR4 (TLR4), CD14 (CD14), Endocytosis (内吞作用), Macrophage (巨噬细胞), BMDM (BMDM), Innate immunity (先天性免疫), LPS (LPS)

Background

Innate immune cells, including macrophages and dendritic cells, employ a variety of pattern recognition receptors (PRRs) to survey their environments for danger- and pathogen-associated molecular patterns. Trafficking and signaling of PRRs from various subcellular compartments enables wider immune surveillance and has emerged as an important design principle of innate immunity (Brubaker et al., 2015). The detection of the bacterial endotoxin LPS is highly dependent on TLR4 and its co-receptor CD14. The endocytosis of the TLR4 complex requires CD14 and is essential for LPS-induced macrophage activation (Zanoni et al., 2011; Tan et al., 2015). Endocytosis of TLR4 is essential to activate secondary signaling complexes at the newly-formed ‘signaling endosome,’ which promotes interferon regulatory factor 3-dependent transcription through the signaling adaptor TIR-domain containing adapter-inducing interferon-β (TRIF) (Kagan et al., 2008). TLR4 endocytosis has been observed in macrophages, dendritic cells, and epithelial cells (Roy et al., 2014). Understanding the underlying mechanisms of this critical step in macrophage activation requires a robust and quantitative method to measure LPS-triggered TLR4 endocytosis. Here, we describe a version of a flow cytometry-based method that was initially reported by Kagan and colleagues (Kagan et al., 2008), and used by others, to monitor TLR4 endocytosis. We have used the method recently to study the role of transient receptor potential melastatin-like 7 (TRPM7), an ion channel, in TLR4 endocytosis (Schappe et al., 2018). The experimental logic of this method relies on measuring the loss of TLR4 and CD14 staining at the cell surface after LPS treatment. We stain LPS-treated cells with an anti-TLR4 (or anti-CD14) fluorophore-conjugated antibody without permeabilization. The fluorescence intensity acquired using flow cytometry reports the relative quantity of receptor resident in the plasma membrane (Figure 1). Although specific for TLR4 and CD14, the assay can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.


Figure 1. Schematic of TLR4 and CD14 endocytosis protocol. Experimental workflow described in protocol “Procedure”.

Materials and Reagents

  1. Materials
    1. Pipette tips
    2. 5 ml round, disposable round-bottom tube (FACS Tube) (Corning, Falcon®, catalog number: 352052 )
    3. Aluminum foil (Genesee Scientific, catalog number: 88-101 )
    4. 0.2 μm bottle filter (Thermo Fisher Scientific, NalgeneTM, catalog number: 566-0020 )
    5. 6-well non-treated culture plates (Corning, catalog number: 3736 )
    6. Sterile cell scrapers (Fisher Scientific, FisherbrandTM, catalog number: 08-100-240 )
    7. Sterile individually packaged serological pipette (10 ml) (Greiner Bio One International, catalog number: 607160 )
    8. Sterile individually packaged serological pipette (5 ml) (Greiner Bio One International, catalog number: 606160 )
    9. 1.7 ml microfuge Eppendorf tubes (Genesee Scientific, Olympus Plastics, catalog number: 24-281 )
    10. NuncTM TripleFlaskTM Treated Cell Culture Flasks (Thermo Fisher Scientific, catalog number: 132867 )
    11. Falcon® 50 ml Conical Centrifuge Tube (Corning, catalog number: 352098 )

  2. Cell line
    1. L-929 cells (ATCC, catalog number: CCL-1 )

  3. Reagents
    1. LPS EB-Ultrapure (lipopolysaccharide from E. coli O111:B4, InvivoGen, catalog number: tlrl-3pelps )
    2. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    3. Mouse TruStain fcXTM (anti-CD16/32) (BioLegend, catalog number: 101320 )
    4. TLR4 [anti-mouse CD284] (PE) (clone: SA15-21; isotype: Rat IgG2a, κ) (BioLegend, catalog number: 145404 )
    5. CD14 [anti-mouse] (APC) (clone: Sa2-8; isotype: Rat IgG2a, κ) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 17-0141-81 )
    6. RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
    7. Fetal bovine serum (heat-inactivated), certified, USA origin (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
    8. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    9. HBSS, no calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14170112 )
    10. BSA (Bovine serum albumin) (Roche Molecular Systems, catalog number: 3116956001 )
    11. DMEM, high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 )
    12. BMDM Media (see Recipes)
    13. Culture Media (see Recipes)
    14. Treatment Media (see Recipes)
    15. FACS Buffer (see Recipes)
    16. L929-conditioned media (see Recipes)

Equipment

  1. TC20 Automated cell counter (Bio-Rad Laboratories, catalog number: 1450102 )
  2. Pipet-aid Pipette Controller (Drummond Scientific, catalog number: 4-000-101 )
  3. 4 °C Cold Room
  4. 4 °C Benchtop centrifuge
  5. 37 °C Cell Culture Incubator with CO2 control
  6. Sterile cell culture hood
  7. Flow Cytometer (BD, model: FACSCantoTM II , or equivalent)

Software

  1. GraphPad Prism 7 (Graph Pad Software; La Jolla, CA USA)

Procedure

  1. Day 1, Cell culture
    1. Collect cultured bone marrow-derived macrophages (BMDMs) by gentle scraping. Disperse the cells into a single-cell suspension by repeatedly running the pipetted cell suspension along the test tube walls. Centrifuge cells (400 x g, 5 min, 23 °C), aspirate supernatant, and resuspend cell pellet in BMDM media. Count live cells via trypan blue exclusion assay.
    2. Plate 0.5 x 106 cells/well in a 6-well, non-treated, tissue culture plate. Culture the cells overnight in 2 ml/well of BMDM media. After 16 h of incubation (37 °C, 5% CO2), cells should be adherent and ready for the experiment.
  2. Day 0, Preparation before LPS stimulation
    1. Prepare Culture and Treatment Media as described in Recipes. Warm the LPS-containing treatment media to 37 °C prior to use.
    2. Chill sterile PBS, 1.5 ml Eppendorf tubes, and FACS buffer to 4 °C, prior to use. After LPS treatment, the cells will be collected using these solutions and tubes.
  3. Day 0, LPS stimulation of cells
    1. Aspirate BMDM media and wash 3 x with 3 ml of HBSS (room temperature) to remove dead cells and debris from each well. Add HBSS down the wall of the culture well and gently swirl plate to wash.
    2. Gently add 2 ml of Culture Media to wells labeled “Unstained BMDMs” and “t = 0 min/Untreated” treatment groups.
    3. Gently add 2 ml of Treatment Media to each well by pipetting the media along the side of the wells. Gently swirl the plate to ensure that the media is evenly distributed in the wells.
    4. Incubate at 37 °C for desired time points. Repeat Steps 3a to 3c as necessary for remaining LPS-treatment groups. Stagger the LPS treatment such that all samples are harvested at the same time.
  4. Day 2, Cell collection and antibody staining
    Note: All reagents should be cold and the procedure should be performed at 4 °C (cold room).
    1. Transfer plates treated in Step 3 to 4 °C for 5 min prior to collection – this is required to arrest endocytosis.
    2. Aspirate media from each well. Wash 2 x each with 2 ml of sterile, pre-chilled PBS.
    3. Add 1 ml of sterile, pre-chilled PBS to each well. Gently scrape to detach cells and pipette-mix to disperse the cells into a single cell suspension.
    4. Transfer the cell suspension to 1.5 ml Eppendorf tubes and centrifuge (400 x g, 5 min) to pellet the cells.
    5. After aspirating and discarding the supernatant, resuspend the cell pellet in 50 μl of cold FACS Buffer premixed with TruStain fcXTM antibody (1 μg/ml), for 10 min.
    6. Add 50 μl of the 2x-concentrate of antibody (anti-TLR4 or anti-CD14; see Table 1) in FACS Buffer to the cell suspension. Add 50 μl of FACS Buffer to “Unstained BMDMs” samples.

      Table 1. Antibodies used for measuring TLR4 and CD14 endocytosis


    7. Pipette gently to mix and stain for 20 min in the dark.
    8. Add 1 ml of FACS Buffer, collect cells by centrifugation (400 x g, 5 min) and aspirate supernatant to remove excess antibody.
    9. Resuspend the cell pellet in 200 μl of FACS Buffer and transfer the cell suspension to FACS tubes. Keep samples on ice and in the dark (e.g., cover with aluminum foil) prior to measurement by flow cytometry.
    10. Analyze samples via flow cytometry within 1 h.

Data analysis

  1. Flow cytometry analysis
    1. For flow cytometry analysis, collect > 100,000 events for each sample.
    2. For analysis, the events are gated on FSC-A and SSC-A bivariant cytographs; the low FSC-SSC events comprising of dead cells and cellular debris are excluded from analysis. Cells are then gated on FSC-A and FSC-H to gate on single cells.
    3. These cells can then be visualized for the intensity of the antibody stain as a histogram. Fluorescent intensity of the antibody stain on the gated population is recorded as the geometric mean of the cell population, or mean fluorescent intensity (MFI). 
  2. Data analysis for the measurement of TLR4 and CD14 Endocytosis
    1. For data analysis, the MFI of “Unstained cells” can be used for background subtraction from all samples [“Background Subtracted MFI”]. Divide the “background subtracted MFI” value for a given time point by the “Unstimulated [t = 0 min]” sample; “t = 0 min” value should be 1.00. Repeat this for all subsequent experimental samples to determine “Relative % of surface expression” relative to the “Unstimulated [t = 0 min]” sample. All sample values reflect the ratio of MFI from stimulated to unstimulated cells at desired time points.
    2. We convert the “Relative % of surface expression” value to a percentage; Thus, the “Untreated” or “Time = 0 min” sample should equal ‘100% percentage of surface expression’. One expects to see a steady reduction in this value at various time points after LPS stimulation.
    3. Since MFI values are sensitive to variations in flow cytometry calibrations, we recommend that data analysis be confined to each independent experiment and each condition run in technical triplicate. “Percentage of surface expression” should be reproducible across independent experiments and therefore amenable to statistical analysis of multiple experiments. Results from a typical experiment are shown in Figure 2. The original data presentation and additional information are available in our manuscript, which originally utilized the protocol described herein PubMed.


      Figure 2. TLR4 and CD14 receptor endocytosis data analysis and suggested presentation. A. “Data analysis” calculation described as a formula. B. Characteristic TLR4 and CD14 endocytosis measured over time in bone-marrow derived macrophages. Data was modified from its original presentation in Schappe et al., 2018 with author permission.

  3. For experimental and statistical analysis, we use GraphPad Prism. To compare two experimental groups, we use a Student’s t-test. For comparison of three or more data groups, other statistical analysis, such as a one-way ANOVA, are necessary.

Notes

  1. The murine macrophage RAW 264.7 cell line also exhibits characteristic TLR4 endocytosis–it can be used to establish the method and for experiments.
  2. Although non-treated culture plates may permit detachment with trypsin, the enzymatic detachment may alter macrophage epitope expression at the plasma membrane.
  3. Performing “Procedure” Step 4 in a walk-in 4 °C cold room greatly improves the quality of data. Although chilling materials and reagents on ice may be convenient, variations in temperature between the ice, samples, and ambient laboratory air may inadvertently warm samples above 4 °C, thereby permitting endocytosis to proceed.
  4. Although procedural steps after “Procedure” Step 3d are not performed under sterile conditions, using sterile reagents minimizes inadvertent contamination with ligands that may promote TLR4 or CD14 endocytosis.
  5. Although spectrally non-overlapping fluorophores are available, we advise staining with a single anti-TLR4 or CD14 antibody for each experiment.
  6. With careful spectral consideration, fluorescent live/dead dyes can be included in this assay to enrich for live cell populations. If the experimenter includes these dyes, we advise that only spectrally-compatible nucleic acid binding dyes, which can rapidly label dead cells during the final suspension be used. Some viability dyes and staining methods (such as “live/dead fixable dyes” or Annexin V-based staining kits) require additional staining steps that may compromise the time and temperature-sensitivity of this assay.
  7. Titration of antibodies, including new batches of the same antibody clone, is essential. Although recommendations are provided in Table 1, improper staining will limit signal-to-noise ratio (SNR) in the assay and lower data quality in terms of sensitivity and consistency.
  8. Avoid sample groups larger than 24 samples to minimize sample processing time prior to flow cytometry analysis.
  9. The majority of our data were collected on the BD FACSCanto II flow cytometer.

Recipes

  1. BMDM Media
    1. RPMI 1640 + 10% FBS + 20% L929-conditioned media
    2. Store at 4 °C for up to 1 month
  2. Culture Media
    1. RPMI 1640 + 10% FBS
    2. Store at 4 °C for up to 1 month
  3. Treatment Media
    1. RPMI 1640 + 10% FBS + 1 μg/ml LPS
    2. Prepare fresh for each experiment
  4. FACS Buffer
    1. PBS + 1% BSA
    2. Sterile filter buffer through 0.22 μm filter prior to use
    3. Store at 4 °C for up to 1 week
  5. L929-conditioned Media
    Note: Generated from the culture of L-929 cells (available from ATCC). Cells are passaged according to the vendor’s instructions and cultured in DMEM, high glucose + 10% FBS.
    1. To generate L929-conditioned media, add 150 ml of DMEM, high glucose + 10% FBS to a T-150 TripleFlask. Add 0.72 x 106 L-929 cells and carefully mix by equilibrating the media volume at the hole in the corner of the flask. Culture for 7 days at 37 °C, 5% CO2
    2. On Day 7, collect media, sterile filter through a 0.22 μm filter into a flask, and store at -20 °C [“Week 1 media”]; add 150 ml of DMEM, high glucose + 10% FBS to TripleFlask to replace collected media
    3. On Day 14, collect media from the flask and sterile filter through a 0.22 μm filter [“Week 2 media”]. Thaw Week 1 media at 23 °C
    4. Combine Week 1 and Week 2 media and aliquot into 50 ml tubes. L929-conditioned media is stored at -20 °C for up to 6 months

Acknowledgments

We would like to thank Dr. Jonathan Kagan (Boston Children’s Hospital) and colleagues whose protocol (cited herein) we adapted for our research. We also would like to thank members of the Desai laboratory for their assistance in editing this manuscript. We also thank the UVA Flow Cytometry Core and Carter Immunology Center Flow Cytometry Core. We appreciate our funding that supported this work: grants GM108989 (BND) and 5T32GM007055-41 (MSS) from the National Institutes of Health.
The authors declare that they have no conflicts of interest to report.

References

  1. Brubaker, S. W., Bonham, K. S., Zanoni, I. and Kagan, J. C. (2015). Innate immune pattern recognition: a cell biological perspective. Annu Rev Immunol 33: 257-290.
  2. Kagan, J. C., Su, T., Horng, T., Chow, A., Akira, S. and Medzhitov, R. (2008). TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-β. Nat Immunol 9(4): 361-368.
  3. Roy, S., Karmakar, M. and Pearlman, E. (2014). CD14 mediates Toll-like receptor 4 (TLR4) endocytosis and spleen tyrosine kinase (Syk) and interferon regulatory transcription factor 3 (IRF3) activation in epithelial cells and impairs neutrophil infiltration and Pseudomonas aeruginosa killing in vivo. J Biol Chem 289(2): 1174-1182.
  4. Schappe, M. S., Szteyn, K., Stremska, M. E., Mendu, S. K., Downs, T. K., Seegren, P. V., Mahoney, M. A., Dixit, S., Krupa, J. K., Stipes, E. J., Rogers, J. S., Adamson, S. E., Leitinger, N. and Desai, B. N. (2018). Chanzyme TRPM7 mediates the Ca2+ influx essential for lipopolysaccharide-induced toll-like receptor 4 endocytosis and macrophage activation. Immunity 48(1): 59-74 e55.
  5. Tan, Y., Zanoni, I., Cullen, T. W., Goodman, A. L. and Kagan, J. C. (2015). Mechanisms of toll-like receptor 4 endocytosis reveal a common immune-evasion strategy used by pathogenic and commensal bacteria. Immunity 43(5): 909-922.
  6. Zanoni, I., Ostuni, R., Marek, L. R., Barresi, S., Barbalat, R., Barton, G. M., Granucci, F. and Kagan, J. C. (2011). CD14 controls the LPS-induced endocytosis of Toll-like receptor 4. Cell 147(4): 868-880.

简介

在识别细胞外细菌脂多糖(LPS)后,Toll样受体4(TLR4)-CD14信号传导复合物启动两种不同的信号传导途径 - 一种来自质膜,另一种来自信号传导内体(Kagan 等。,2008)。因此,了解TLR4信号转导的早期阶段需要一种稳健且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用,这是LPS检测中最早发生的事件之一。在这里,我们描述了一种基于流式细胞术的方法,我们最近用它来研究离子通道TRPM7在TLR4内吞作用中的作用(Schappe et al。,2018)。该测定依赖于用LPS刺激细胞并使用流式细胞术在不同时间点测量TLR4(或CD14)的细胞表面水平。尽管我们详细描述了来自鼠骨髓来源的巨噬细胞的TLR4和CD14的方法,但它可以很容易地适应于在各种其他信号传导环境中评估受体内吞作用。

【背景】先天免疫细胞,包括巨噬细胞和树突细胞,使用各种模式识别受体(PRR)来调查其环境中的危险和病原体相关分子模式。来自各种亚细胞区室的PRR的贩运和信号传导实现了更广泛的免疫监视,并且已成为先天免疫的重要设计原则(Brubaker et al。,2015)。细菌内毒素LPS的检测高度依赖于TLR4及其共同受体CD14。 TLR4复合物的内吞作用需要CD14,并且对于LPS诱导的巨噬细胞活化是必需的(Zanoni 等人,2011; Tan 等人,2015)。 TLR4的内吞作用对于在新形成的'信号传导内体'中激活二级信号传导复合物是必需的,其通过信号传导适配子TIR结构域诱导干扰素调节因子3依赖性转录 - 诱导干扰素-β(TRIF)(Kagan 等人,2008)。已经在巨噬细胞,树突细胞和上皮细胞中观察到TLR4内吞作用(Roy 等人,,2014)。了解巨噬细胞活化中这一关键步骤的潜在机制需要一种稳健的定量方法来测量LPS触发的TLR4内吞作用。在这里,我们描述了一种基于流式细胞术的方法,该方法最初由Kagan及其同事报告(Kagan et al。,2008),并被其他人用于监测TLR4内吞作用。我们最近使用该方法来研究瞬时受体电位melastatin-like 7(TRPM7)(一种离子通道)在TLR4内吞作用中的作用(Schappe et al。,2018)。该方法的实验逻辑依赖于测量LPS处理后细胞表面TLR4和CD14染色的丧失。我们用抗TLR4(或抗CD14)荧光团 - 缀合的抗体染色LPS处理的细胞而不透化。使用流式细胞术获得的荧光强度报告了位于质膜中的受体的相对量(图1)。虽然特异于TLR4和CD14,但是该测定可以容易地适应于在各种其他信号传导环境中评估受体内吞作用。


图1. TLR4和CD14内吞作用方案的示意图。方案“程序”中描述的实验工作流程。

关键字:Toll-类受体, TLR, TLR4, CD14, 内吞作用, 巨噬细胞, BMDM, 先天性免疫, LPS

材料和试剂

  1. 物料
    1. 移液器吸头
    2. 5毫升圆形一次性圆底管(FACS管)(Corning,Falcon ®,目录号:352052)
    3. 铝箔(Genesee Scientific,目录号:88-101)
    4. 0.2μm瓶式过滤器(Thermo Fisher Scientific,Nalgene TM ,目录号:566-0020)
    5. 6孔未处理的培养板(Corning,目录号:3736)
    6. 无菌细胞刮刀(Fisher Scientific,Fisherbrand TM ,目录号:08-100-240)
    7. 无菌单独包装的血清移液管(10 ml)(Greiner Bio One International,目录号:607160)
    8. 无菌单独包装的血清移液管(5 ml)(Greiner Bio One International,目录号:606160)
    9. 1.7毫升微量离心机Eppendorf管(Genesee Scientific,Olympus Plastics,目录号:24-281)
    10. Nunc TM TripleFlask TM 处理细胞培养瓶(Thermo Fisher Scientific,目录号:132867)
    11. Falcon ® 50 ml锥形离心管(Corning,目录号:352098)

  2. 细胞系
    1. L-929细胞(ATCC,目录号:CCL-1)

  3. 试剂
    1. LPS EB-Ultrapure(来自大肠杆菌的脂多糖 O111:B4,InvivoGen,目录号:tlrl-3pelps)
    2. PBS(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
    3. Mouse TruStain fcX TM (抗CD16 / 32)(BioLegend,目录号:101320)
    4. TLR4 [抗小鼠CD284](PE)(克隆:SA15-21;同种型:大鼠IgG2a,κ)(BioLegend,目录号:145404)
    5. CD14 [抗小鼠](APC)(克隆:Sa2-8;同种型:大鼠IgG2a,κ)(Thermo Fisher Scientific,eBioscience TM ,目录号:17-0141-81)
    6. RPMI 1640(Thermo Fisher Scientific,Gibco TM ,目录号:11875093)
    7. 胎牛血清(热灭活),经认证,美国原产地(Thermo Fisher Scientific,Gibco TM ,目录号:10082147)
    8. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    9. HBSS,无钙,无镁(赛默飞世尔科技,Gibco TM ,目录号:14170112)
    10. BSA(牛血清白蛋白)(Roche Molecular Systems,目录号:3116956001)
    11. DMEM,高糖(Thermo Fisher Scientific,Gibco TM ,目录号:11965092)
    12. BMDM媒体(见食谱)
    13. 文化传媒(见食谱)
    14. 治疗媒体(见食谱)
    15. FACS缓冲液(见食谱)
    16. L929条件培养基(见食谱)

设备

  1. TC20自动细胞计数器(Bio-Rad Laboratories,目录号:1450102)
  2. 移液器移液器控制器(Drummond Scientific,目录号:4-000-101)
  3. 4°C冷室
  4. 4°C台式离心机
  5. 37°C细胞培养培养箱,CO 2 对照
  6. 无菌细胞培养罩
  7. 流式细胞仪(BD,型号:FACSCanto TM II,或等效物)

软件

  1. GraphPad Prism 7(Graph Pad Software; La Jolla,CA USA)

程序

  1. 第1天,细胞培养
    1. 通过温和刮擦收集培养的骨髓衍生的巨噬细胞(BMDM)。通过沿试管壁反复运行移液细胞悬液,将细胞分散成单细胞悬液。离心细胞(400 x g ,5分钟,23℃),吸出上清液,并在BMDM培养基中重悬细胞沉淀。通过台盼蓝排除试验计数活细胞。
    2. 在6孔未处理的组织培养板中平板0.5×10 6个细胞/孔。将细胞在2ml /孔的BMDM培养基中培养过夜。孵育16小时后(37℃,5%CO 2 ),细胞应粘附并准备好进行实验。
  2. 第0天,LPS刺激前的准备
    1. 按照食谱中的描述准备培养和处理培养基。使用前将含LPS的处理介质温热至37°C。
    2. 在使用前,将无菌PBS,1.5ml Eppendorf管和FACS缓冲液冷却至4℃。在LPS处理后,使用这些溶液和管收集细胞。
  3. 第0天,LPS刺激细胞
    1. 吸出BMDM培养基并用3ml HBSS(室温)洗涤3次以除去每个孔中的死细胞和碎片。在培养孔壁上加入HBSS,轻轻旋转盘子洗净。
    2. 向标记为“未染色BMDM”和“ t = 0分钟/未处理”治疗组的孔中轻轻加入2 ml培养基。
    3. 通过沿着孔的侧面移液培养基,向每个孔中轻轻加入2ml处理培养基。轻轻旋转平板,确保介质均匀分布在孔中。
    4. 在37°C孵育所需的时间点。对于剩余的LPS处理组,根据需要重复步骤3a至3c。错开LPS处理使得所有样品同时收获。
  4. 第2天,细胞收集和抗体染色
    注意:所有试剂都应冷却,程序应在4°C(冷室)下进行。
    1. 在收集前将步骤3中处理的转移板加热至4℃5分钟 - 这是阻止内吞作用所必需的。
    2. 从每个井中吸出培养基。用2ml无菌预冷的PBS洗涤2次。
    3. 每孔加入1ml无菌预冷的PBS。轻轻刮去分离细胞和移液管混合物,将细胞分散成单细胞悬液。
    4. 将细胞悬浮液转移至1.5ml Eppendorf管中并离心(400 x g ,5分钟)以沉淀细胞。
    5. 抽吸并弃去上清液后,将细胞沉淀重悬于50μl预混有TruStain fcX TM 抗体(1μg/ ml)的冷FACS缓冲液中10分钟。
    6. 在FACS缓冲液中将50μl2x浓度的抗体(抗TLR4或抗CD14;参见表1)加入细胞悬浮液中。向“未染色的BMDM”样品中加入50μlFACS缓冲液。

      表1.用于测量TLR4和CD14内吞作用的抗体


    7. 轻轻吸取混合物,在黑暗中染色20分钟。
    8. 加入1 ml FACS Buffer,离心收集细胞(400 x g ,5 min),吸出上清液,去除多余的抗体。
    9. 将细胞沉淀重悬于200μlFACS缓冲液中,并将细胞悬浮液转移至FACS管中。在通过流式细胞仪测量之前,将样品保存在冰上和黑暗中(例如,用铝箔覆盖)。
    10. 在1小时内通过流式细胞术分析样品。

数据分析

  1. 流式细胞术分析
    1. 对于流式细胞术分析,收集>每个样本100,000个事件。
    2. 为了分析,事件是在FSC-A和SSC-A双变量细胞仪上进行门控的;从分析中排除包含死细胞和细胞碎片的低FSC-SSC事件。然后在FSC-A和FSC-H上对细胞进行门控以在单个细胞上进行门控。
    3. 然后可以将这些细胞作为直方图显示抗体染色的强度。门控群体上抗体染色的荧光强度记录为细胞群的几何平均值,或平均荧光强度(MFI)。 
  2. 用于测量TLR4和CD14胞吞作用的数据分析
    1. 对于数据分析,“未染色细胞”的MFI可用于从所有样品中减去背景[“背景减去MFI”]。将给定时间点的“背景扣除MFI”值除以“未刺激[t = 0 min]”样本; “t = 0 min”值应为1.00。对所有后续实验样品重复此操作以确定相对于“未刺激的[t = 0分钟]”样品的“表面表达的相对%”。所有样本值反映了在所需时间点受刺激细胞和未受刺激细胞的MFI比率。
    2. 我们将“表面表达式的相对百分比”值转换为百分比;因此,“未处理”或“时间= 0分钟”样品应该等于“表面表达的100%百分比”。人们预计在LPS刺激后的不同时间点,该值会稳定下降。
    3. 由于MFI值对流式细胞仪校准的变化敏感,我们建议将数据分析限制在每个独立实验中,并且每个条件以技术三份重复进行。 “表面表达的百分比”应该在独立实验中可重复,因此适合于多个实验的统计分析。典型实验的结果如图2所示。原始数据表示和其他信息可在我们的手稿中找到,该手稿最初使用了此处描述的协议 PubMed 。


      图2. TLR4和CD14受体内吞作用数据分析和建议的表达。 A.“数据分析”计算描述为公式。 B.在骨髓衍生的巨噬细胞中随时间测量的特征性TLR4和CD14胞吞作用。根据作者的许可,数据在Schappe et al。,2018年的原始演示文稿中进行了修改。

  3. 对于实验和统计分析,我们使用GraphPad Prism。为了比较两个实验组,我们使用学生的 t - 测试。为了比较三个或更多数据组,需要其他统计分析,例如单向ANOVA。

笔记

  1. 小鼠巨噬细胞RAW 264.7细胞系也表现出特征性的TLR4内吞作用 - 它可用于建立方法和实验。
  2. 尽管未经处理的培养板可能允许与胰蛋白酶分离,但酶促分离可能会改变质膜上巨噬细胞表位的表达。
  3. 执行“程序”步骤4在步入式4°C冷室中大大提高了数据质量。尽管在冰上冷却材料和试剂可能很方便,但冰,样品和环境实验室空气之间的温度变化可能会无意中将样品加热到4°C以上,从而允许内吞作用进行。
  4. 虽然“程序”步骤3d之后的程序步骤不在无菌条件下进行,但使用无菌试剂可最大限度地减少无意中污染配体,从而促进TLR4或CD14内吞作用。
  5. 虽然可以获得光谱上不重叠的荧光团,但我们建议每次实验用单一的抗TLR4或CD14抗体染色。
  6. 通过仔细的光谱考虑,荧光活/死染料可以包括在该测定中以富集活细胞群。如果实验者包括这些染料,我们建议只使用光谱兼容的核酸结合染料,它可以在最终悬浮期间快速标记死细胞。一些活力染料和染色方法(例如“活/死固定染料”或基于膜联蛋白V的染色试剂盒)需要额外的染色步骤,这可能会影响该测定的时间和温度敏感性。
  7. 抗体的滴定,包括同一抗体克隆的新批次,是必不可少的。虽然表1中提供了建议,但不正确的染色会限制测定中的信噪比(SNR),并且在灵敏度和一致性方面会降低数据质量。
  8. 避免样本组大于24个样本,以便在流式细胞术分析之前最大限度地缩短样本处理时间。
  9. 我们的大部分数据都是在BD FACSCanto II流式细胞仪上收集的。

食谱

  1. BMDM媒体
    1. RPMI 1640 + 10%FBS + 20%L929条件培养基
    2. 在4°C下储存长达1个月
  2. 文化传媒
    1. RPMI 1640 + 10%FBS
    2. 在4°C下储存长达1个月
  3. 治疗媒体
    1. RPMI 1640 + 10%FBS +1μg/ ml LPS
    2. 为每个实验准备新鲜的
  4. FACS缓冲液
    1. PBS + 1%BSA
    2. 使用前无菌过滤缓冲液通过0.22μm过滤器
    3. 在4°C下储存长达1周
  5. L929条件媒体
    注意:由L-929细胞培养物(可从ATCC获得)产生。根据供应商的说明传代细胞,并在DMEM,高葡萄糖+ 10%FBS中培养。
    1. 为了产生L929条件培养基,向T-150 TripleFlask中加入150ml DMEM,高葡萄糖+ 10%FBS。加入0.72×10 6个 L-929细胞,并通过平衡烧瓶角部孔处的培养基体积小心地混合。在37℃,5%CO 2 培养7天
    2. 在第7天,将培养基,无菌过滤器通过0.22μm过滤器收集到烧瓶中,并储存在-20°C [“第1周培养基”];向TripleFlask中加入150毫升DMEM,高葡萄糖+ 10%FBS以替换收集的培养基
    3. 在第14天,从烧瓶和无菌过滤器通过0.22μm过滤器[“第2周培养基”]收集培养基。在23°C解冻第1周媒体
    4. 将第1周和第2周的培养基和等分试样合并到50ml管中。将L929条件培养基在-20°C下储存长达6个月

致谢

我们要感谢Jonathan Kagan博士(波士顿儿童医院)及其同事,他们的协议(在此引用)我们为我们的研究进行了调整。我们还要感谢Desai实验室的成员协助编辑本手稿。我们还要感谢UVA流式细胞仪核心和卡特免疫中心流式细胞仪核心。我们感谢我们的资助支持这项工作:从美国国立卫生研究院获得GM108989(BND)和5T32GM007055-41(MSS)。
作者声明他们没有利益冲突报告。

参考

  1. Brubaker,S.W.,Bonham,K.S.,Zanoni,I。和Kagan,J。C.(2015)。 先天免疫模式识别:细胞生物学观点。 A nnu Rev Immun o l 33:257-290。
  2. Kagan,J。C.,Su,T.,Horng,T.,Chow,A.,Akira,S。和Medzhitov,R。(2008)。 TRAM将Toll样受体4的内吞作用与干扰素-β的诱导结合起来。 Nat Immunol 9(4):361-368。
  3. Roy,S.,Karmakar,M。和Pearlman,E。(2014)。 CD14介导Toll样受体4(TLR4)内吞作用和脾酪氨酸激酶(Syk)和干扰素调节转录因子3(IRF3)在上皮细胞中激活并损害中性粒细胞浸润和铜绿假单胞菌在体内杀死。 J Biol Che m 289(2):1174-1182。
  4. Schappe,MS,Szteyn,K.,Stremska,ME,Mendu,SK,Downs,TK,Seegren,PV,Mahoney,MA,Dixit,S.,Krupa,JK,Stipes,EJ,Rogers,JS,Adamson,SE, Leitinger,N。和Desai,BN(2018)。 Chanzyme TRPM7介导脂多糖诱导的收费所必需的Ca 2 + 流入类似受体4内吞作用和巨噬细胞激活。 免疫 48(1):59-74 e55。
  5. Tan,Y.,Zanoni,I.,Cullen,T.W。,Goodman,A。L.和Kagan,J。C.(2015)。 Toll样受体4内吞作用的机制揭示了致病菌和共生细菌使用的常见免疫逃避策略。 我 mmunity 43(5):909-922。
  6. Zanoni,I.,Ostuni,R.,Marek,L.R.,Barresi,S.,Barbalat,R.,Barton,G.M.,Granucci,F。和Kagan,J。C.(2011)。 CD14控制LPS诱导的Toll样受体4的内吞作用。 Cell 147(4):868-880。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Schappe, M. S. and Desai, B. N. (2018). Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry. Bio-protocol 8(14): e2926. DOI: 10.21769/BioProtoc.2926.
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