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Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining
淋巴细胞分离、Th17细胞分化、激活和染色   

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Abstract

In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.

Keywords: Th17(Th17), IL-17(IL-17), FACS(FACS)

Background

T cells are critical to mediate host defense against bacteria, viruses and fungi as well as commensal (Kumar et al., 2016). T cells can be further subdivided into T helper (Th1), Th2 and Th17 subsets based on their ability to generate specific cytokines. Naive T cells can be differentiated into specific T cell subsets in in vitro culture in response to specific cytokine stimulation. In vitro generated Th1, Th2 and Th17 cells have helped us to understand the molecular mechanism of their differentiation and their effector functions. Here, we have described a basic protocol for Th17 cell generation.

Materials and Reagents

  1. 96-well tissue culture plate (CELLTREAT Scientific Products, catalog number: 229196 )
  2. Falcon® 70 μm cell strainers (Corning, Falcon®, catalog number: 352350 )
  3. 50 ml conical tube (Denville Scientific, catalog number: C1056 )
  4. 1 ml syringe with cap (BD, catalog number: 305217 )
  5. 15 ml conical tube (Denville Scientific, catalog number: C1018-P )
  6. 96-well round (U) bottom plate (CELLTREAT Scientific Products, catalog number: 229190 )
  7. C57BL\6 mice (Taconic Biosciences, catalog number: B6-F )
  8. Coating antibodies: anti-mouse CD28 (Affymetrix, eBioscience, catalog number: 14-0281 ), anti-mouse CD3e (Affymetrix, eBioscience, catalog number: 14-0033 )
  9. ELISA coating buffer (Biolegend, catalog number: 421701 )
  10. EasySepTM buffer (STEMCELL Technologies, catalog number: 20144 ) or PBS containing 2% fetal bovine serum (FBS) with 1 mM EDTA
  11. EasySepTM Mouse Naïve CD4+ T Cell Isolation Kit (STEMCELL Technologies, catalog number: 19765 )
  12. Staining antibodies:
    1.  anti-mouse CD3-eFlour 450 (Affymetrix, eBioscience, catalog number: 48-0032 )
    2.  anti-mouse CD4-Alexa Flour 700 (Affymetrix, eBioscience, catalog number: 56-0041 )
    3.  anti-mouse IL-22-APC (Affymetrix, eBioscience, catalog number: 17-7222 )
    4.  anti-mouse IL-17A-PE (Affymetrix, eBioscience, catalog number: 12-7177 )
    5.  anti-mouse CD62L-FITC (Affymetrix, eBioscience, catalog number: 11-0621 )
  13. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  14. Ionomycin (Sigma-Aldrich, catalog number: I0634 )
  15. Fixation/Permeabilization Solution Kit with BD GolgiStopTM (BD, catalog number: 554715 )
  16. Cytofix/Cytoperm Plus Kit (with BD GolgiStopTM) (BD, catalog number: 554715 )
  17. Iscove’s modified Dulbecco’s medium (IMDM) cell culture medium + glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 31980-030 )
  18. HyCloneTM fetal bovine serum (U.S.), characterized FBS (GE Healthcare, catalog number: SH30071.03 )
  19. HyCloneTM penicillin-streptomycin 100x solution (GE Healthcare, catalog number: SV30010 )
  20. Recombinant porcine TGF-β1 (R&D Systems, catalog number: 101-B1 )
  21. Recombinant mouse IL-6 (R&D Systems, catalog number: 406-ML )
  22. Recombinant mouse IL-23 (R&D Systems, catalog number: 1887-ML )
  23. Anti-mouse IFNγ (R&D Systems, catalog number: MAB485 )
  24. Anti-mouse IL-4 (R&D Systems, catalog number: MAB404 )
  25. Phosphate buffer saline (PBS) (Boston BioProduct, catalog number: BM220S )
  26. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3059 )
  27. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  28. Complete IMDM medium (see Recipes)
  29. 2x Th17 differentiation condition medium (see Recipes)
  30. FACS buffer (see Recipes)

Equipment

  1. Tissue culture incubator (NuAire, model: LabGard Class II type A2 )
  2. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall Legend XFR )
  3. BD LSRII Flow cytometer - BD
  4. Scientific Industries Vortex Genie2 (Stellar Scientific, catalog number: SI-236 )
  5. EasyEightTM EasySepTM magnet (STEMCELL Technologies, catalog number: 18103 )

Software

  1. FACS Diva or Flow Jo software

Procedure

  1. Lymphocyte isolation
    1. Coat desired number of wells in a 96-well plate with 50 μl of anti-mouse CD3 (2.5 μg/ml) and anti-mouse CD28 (2.0 μg/ml) for overnight at 4 °C. We used 1x ELISA coating buffer for antibodies dilution and coating. We washed plate once with 1x PBS before adding the cells.
    2. Next day, euthanize a C57BL\6 mice and harvest spleen.
    3. Place 70 micron cell strainer on 50 ml conical tube.
      Place spleen on the cell strainer and homogenize/disrupt using 1 ml syringe cap. Syringe cap is placed on top of the spleen and rotate handle with hand to crush/homogenize the tissue. Add 1 ml EasySep buffer in cell strainer to facilitate homogenization. Collect flow through (contains cells) and add additional 9 ml EasySep buffer directly into the centrifuge tube. Centrifuge 300 x g for 10 min at 4 °C. Discard supernatant and re-suspend cells pellet with 1 ml EasySep buffer. Count cells (RBC lysis step is not required) and add 1 ml EasySep buffer/1 x 108 cells. Transfer cells into a 15 ml centrifuge tube.
      Follow EasySep mouse CD4+CD62L+ naïve T cells isolation protocol.
      Steps for naïve CD4+CD62L+ T cell isolation as per the EasySepTM kit
      Notes:
      1. The total number of cells in a naïve mouse spleen is (~1 x 108) without RBC lyses. We usually get 5-7 million naïve CD4 T cells after the purification steps.
      2. Make sure the purity of isolated CD4+CD62L+ T cells is above 95% after enrichment (see Figure 1). Take small aliquots of purified cells (5,000-10,000 cells) and stain with anti-CD4-Alexa Flour 700 (1:100) and anti-CD62L-FITC (1:100) in ice cold FACS buffer for 30 min at 4 °C. After washing with FACS buffer check purity using flow cytometer by gating for CD4+CD62L+ T cells.
    4. Count the enriched cells and plate them as follows:
      For a 96-well-plate, add 2 x 105 cells/well in 100 μl complete IMDM medium (see Recipes).

  2. Th17 cell differentiation
    1. Prepare 2x Th17 differentiation condition medium (see Recipes)
    2. Add 100 μl 2x Th17 differentiation condition medium to each well.
    3. Culture for 3 additional days at 37 °C and 5% CO2 in a tissue culture incubator. You will see several T cell clusters on day 2 post differentiation and medium will turn slight yellowish. If medium turned very yellowish then add additional 50 μl complete IMDM medium (see Recipes).


      Figure 1. Naïve CD4 T cells purification and Th17 differentiation. A. Flow cytometry dot plot gate shows spleen lymphocytes (left), percentage of CD4+ cells (middle) and frequency of purified naïve CD4+CD62L+ T cells. B. Data show IL-17 producing Th17 cells on day 3 post differentiation.

  3. Activation and staining
    1. After day 3 post differentiation, transfer cells into a U-bottom 96-well plate using a multi-channel pipette (gently pipetting up and down to resuspend the cells) and centrifuge at 300 x g for 5 min at room temperature.
      Discard supernatant by flicking plate in a quick single motion. To flick off the supernatant, move the plate upward and bring it straight down. Gently touch plate with the tissue paper to remove drops. Vortex plates using a benchtop Vortex Genie2 or any mini vortex mixer. Follow these steps for washing and decanting the supernatant.
    2. Add 200 μl of complete IMDM medium containing 50 ng/ml PMA, 750 ng/ml ionomycin and GolgiStop (1:1,000) to the cells and incubate for 4 h at 37 °C and 5% CO2 in a tissue culture incubator.
    3. Wash twice with 200 μl ice cold FACS buffer (300 x g, 5 min, 4 °C). Discard supernatant by flicking plate in a quick single motion.
    4. Re-suspend cells in 100 μl ice cold FACS buffer containing anti-mouse CD3-eFluor 450 (1:100), anti-mouse CD4-Alexa Fluor 700 (1:100) and incubate for 30 min at 4 °C (refrigerator).
    5. Wash cells with FACS buffer (300 G, 2 min, 4 °C).
    6. Fix cells with 100 μl BD Cytofix/Cytoperm for 20 min at 4 °C. Discard supernatant by flicking plate in a quick single motion.
    7. Wash twice with 200 μl 1x BD Perm/WashTM buffer (300 x g, 2 min, 4 °C). Discard supernatant by flicking plate in a quick single motion.
    8. Add 100 μl 1x BD Perm/WashTM buffer containing anti-IL-17A-PE (1:100) and anti-IL-22-APC (1:100) antibodies and incubate for 45 min/overnight at 4 °C.
      Instead of surface staining steps, all antibodies including surface (anti-mouse CD3-eFluor 450, anti-mouse CD4-Alexa Fluor 700) and intracellular (anti-mouse IL-17A-PE and anti-mouse IL-22-APC) can be used together in a single step after fixation and Perm/Wash. We have stained cells with intracellular cytokines along with surface antibodies against CD3 and CD4.
    9. Wash cells twice with 200 μl 1x BD Perm/WashTM buffer (300 x g, 5 min, 4 °C). Re-suspend pellet in 200 μl FACS buffer.
    10. Confirm the lineage of Th17 cell by determining the levels of intracellular IL-17A and IL-22 by FACS. A good Th17 differentiation will result into 20-40% IL-17A producing CD4 T cells.

Data analysis

FACS Diva or Flow Jo software can be used to analyze data. Plot a linear FSC versus SSC dot plot and create a gate (P1) to select all cells. Using P1 population plot a linear CD3 versus CD4 dot plot. Majority of differentiated cells will be positive for both CD3 and CD4. CD3+CD4+ double positive population (gate P2) will be analyzed for intracellular IL-17 and IL-22 staining.

Recipes

  1. Complete IMDM medium (Pre-warmed at 37 °C before use)
    IMDM medium + glutamax
    10% FBS
    1% penicillin/streptomycin solution
  2. 2x Th17 differentiation condition medium
    Complete IMDM medium (Pre-warmed at 37 °C) containing:
    10 ng/ml TGF-β1
    80 ng/ml recombinant IL-6
    20 ng/ml recombinant IL-23
    10 μg/ml anti-IFNγ
    10 μg/ml anti-IL-4
  3. FACS buffer
    PBS with
    0.5 % bovine serum albumin (BSA)
    0.05% sodium azide

Acknowledgments

The authors would like to acknowledge support from the following PHS grants: P50HL084932, 5R01HL061271, and R37HL079142 to J.K.K. P.K is supported from Children’s Hospital of Pittsburgh Research Advisory Committee Grant from Children’s Hospital of Pittsburgh of the UPMC Health System.

References

  1. Kumar, P., Monin, L., Castillo, P., Elsegeiny, W., Horne, W., Eddens, T., Vikram, A., Good, M., Schoenborn, A. A., Bibby, K., Montelaro, R. C., Metzger, D. W., Gulati, A. S. and Kolls, J. K. (2016). Intestinal interleukin-17 receptor signaling mediates reciprocal control of the gut microbiota and autoimmune inflammation. Immunity 44(3): 659-671.

简介

Th17(产生IL-17A,IL-17F和IL-22的α,βT辅助细胞)分化已经常规用于功能性T细胞研究。 这里我们描述Th17细胞分化的方法。
关键词: Th17,IL-17,FACS

[背景] T细胞 是介导宿主防御细菌,病毒和真菌以及共生的关键(Kumar等人,2016)。 T细胞可以基于它们产生特异性细胞因子的能力进一步细分为T辅助(Th1),Th2和Th17子集。 幼稚T细胞可以响应于特异性细胞因子刺激在体外培养中分化成特异性T细胞亚群。 体外产生的Th1,Th2和Th17细胞已经帮助我们理解它们的分化和它们的效应子功能的分子机制。 在这里,我们描述了Th17细胞生成的基本协议。

关键字:Th17, IL-17, FACS

材料和试剂

  1. 96孔组织培养板(CELLTREAT Scientific Products,目录号:229196)
  2. 70μl细胞过滤器(Corning,Falcon ,目录号:352350)。
  3. 50ml锥形管(Denville Scientific,目录号:C1056)
  4. 1ml带盖的注射器(BD,目录号:305217)
  5. 15ml锥形管(Denville Scientific,目录号:C1018-P)
  6. 96孔圆(U)底板(CELLTREAT Scientific Products,目录号:229190)
  7. C57BL/6小鼠(Taconic Biosciences,目录号:B6-F)
  8. 包被抗体:抗小鼠CD28(Affymetrix,eBioscience,目录号:14-0281),抗小鼠CD3e(Affymetrix,eBioscience,目录号:14-0033)
  9. ELISA包被缓冲液(Biolegend,目录号:421701)
  10. EasySep TM缓冲液(STEMCELL Technologies,目录号:20144)或含有含有1mM EDTA的2%胎牛血清(FBS)的PBS
  11. EasySep TM 小鼠初级CD4 + T细胞分离试剂盒(STEMCELL Technologies,目录号:19765)
  12. 染色抗体:
    1. <抗小鼠CD3-eFlour 450(Affymetrix,eBioscience,目录号:48-0032)
    2. <抗小鼠CD4-Alexa Flour 700(Affymetrix,eBioscience,目录号:56-0041)
    3. <抗小鼠IL-22-APC(Affymetrix,eBioscience,目录号:17-7222)
    4. <抗小鼠IL-17A-PE(Affymetrix,eBioscience,目录号:12-7177)
    5. <抗小鼠CD62L-FITC(Affymetrix,eBioscience,目录号:11-0621)
  13. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:79346)
  14. 离子霉素(Sigma-Aldrich,目录号:I0634)
  15. 使用BD GolgiStop TM (BD,目录号:554715)的固定/透化溶液套件
  16. Cytofix/Cytoperm Plus Kit(具有BD GolgiStop TM )(BD,目录号:554715)
  17. Iscove's改良的Dulbecco's培养基(IMDM)细胞培养基+ glutamax(Thermo Fisher Scientific,Gibco TM,目录号:31980-030)
  18. HyClone TM胎牛血清(美国),表征为FBS(GE Healthcare,目录号:SH30071.03)
  19. HyClone 青霉素 - 链霉素100x溶液(GE Healthcare,目录号:SV30010)
  20. 重组猪TGF-β1(R& D Systems,目录号:101-B1)
  21. 重组小鼠IL-6(R& D Systems,目录号:406-ML)
  22. 重组小鼠IL-23(R& D Systems,目录号:1887-ML)
  23. 抗小鼠IFNγγ(R& D Systems,目录号:MAB485)
  24. 抗小鼠IL-4(R& D Systems,目录号:MAB404)
  25. 磷酸盐缓冲盐水(PBS)(Boston BioProduct,目录号:BM220S)
  26. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3059)
  27. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  28. 完成IMDM媒体(请参阅配方)
  29. 2x Th17分化条件培养基(参见配方)
  30. FACS缓冲区(参见配方)

设备

  1. 组织培养箱(NuAire,型号:LabGard Class II type A2)
  2. 离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Sorvall Legend XFR)
  3. BD LSRII流式细胞仪 - BD
  4. 科学工业Vortex Genie2(Stellar Scientific,目录号:SI-236)
  5. EasyEight TM EasySep TM 磁铁(STEMCELL Technologies,目录号:18103)

软件

  1. FACS Diva或Flow Jo软件

程序

  1. 淋巴细胞分离
    1. 在4℃下用50μl抗小鼠CD3(2.5μg/ml)和抗小鼠CD28(2.0μg/ml)在96孔板中涂布所需孔数目过夜。我们使用1x ELISA包被缓冲液进行抗体稀释和包被。我们用1x PBS洗板一次,然后加入细胞。
    2. 第二天,安乐死C57BL \ 6小鼠,收获脾脏
    3. 将70微米的细胞过滤器放在50ml锥形管上 将脾脏放置在细胞滤纸上,并使用1ml注射器盖匀浆/破碎。将注射器盖放置在脾的上部,并用手旋转手柄以压碎/匀化组织。在细胞过滤器中加入1ml EasySep缓冲液以促进匀浆。收集流过(包含细胞),并直接添加9毫升EasySep缓冲液到离心管中。在4℃下离心300×10 10分钟。弃去上清液并用1ml EasySep缓冲液重悬细胞沉淀。计数细胞(不需要RBC裂解步骤),并加入1ml EasySep缓冲液/1×10 8个细胞。转移细胞入15ml离心管。
      按照EasySep小鼠CD4 + CD62L + 初始T细胞分离方案。
      按照 + CD62L + T细胞分离的步骤media/files/pis/28098-PIS_1_0_1.pdf?_ga = 1.200482282.511929587.1455301590"target ="_ blank"> EasySep TM kit
      注意:
      1. 初始小鼠脾脏中的细胞总数是(〜1×10 8个),没有RBC裂解。我们通常在纯化步骤后获得5-7百万个初始CD4 T细胞。
      2. 确保富集后分离的CD4 + T细胞的纯度高于95%(参见图1)。取小份等分的纯化细胞(5,000-10,000个细胞),并在冰冷的FACS缓冲液中在4℃下用抗CD4-Alexa Flour 700(1:100)和抗CD62L-FITC(1:100)染色30分钟。在用FACS缓冲液检查纯度后,使用流式细胞仪通过门控CD4 + CD62L + T细胞洗涤。
    4. 计数富集的细胞,并如下铺平:
      对于96孔板,在100μl完全IMDM培养基中添加2×10 5个细胞/孔(参见Recipes)。

  2. Th17细胞分化
    1. 准备2x Th17分化条件培养基(参见配方)
    2. 每孔加入100μl2x Th17分化条件培养基。
    3. 在37℃和5%CO 2下在组织培养箱中再培养3天。在分化后第2天将看到几个T细胞簇,培养基将变为轻微黄色。如果介质变得非常淡黄色,那么添加额外50ul完整的IMDM介质(见配方)。


      图1.初始CD4T细胞纯化和Th17分化 A.流式细胞术点图显示脾淋巴细胞(左),CD4 +细胞(中)和频率的百分比的纯化的初始CD4 + CD62L + sup/+ T细胞。 B.数据显示分化后第3天产生IL-17的Th17细胞
  3. 活化和染色
    1. 在分化后第3天后,使用多通道移液管将细胞转移到U底96孔板中(轻轻地上下吹打以重悬细胞),并在300×g离心5分钟室内温度。
      通过快速单一动作甩板消除上清液。要甩掉上清液,将平板向上移动并将平直向下。用棉纸轻轻地触摸板以去除液滴。涡旋板使用台式涡旋Genie2或任何微型涡流混合器。按照这些步骤进行洗涤和倾析上清液。
    2. 向细胞中加入200μl含有50ng/ml PMA,750ng/ml离子霉素和GolgiStop(1:1000)的完全IMDM培养基,并在37℃和5%CO 2中孵育4小时,在组织培养孵化器中
    3. 用200μl冰冷的FACS缓冲液(300×g,5分钟,4℃)洗涤两次。通过快速单次移动甩板消除上清液。
    4. 将细胞重悬在含有抗小鼠CD3-eFluor 450(1:100),抗小鼠CD4-Alexa Fluor 700(1:100)的100μl冰冷FACS缓冲液中并在4℃(冰箱)孵育30分钟, 。
    5. 用FACS缓冲液(300G,2分钟,4℃)洗涤细胞
    6. 修复细胞与100微升BD Cytofix/Cytoperm在4℃下20分钟。通过快速单次移动甩板消除上清液。
    7. 用200μl1x BD Perm/Wash TM缓冲液(300×g,2分钟,4℃)洗涤两次。通过快速单次移动甩板消除上清液。
    8. 加入100μl含有抗IL-17A-PE(1:100)和抗IL-22-APC(1:100)抗体的1x BD Perm/Wash 缓冲液,并孵育45分钟/在4℃过夜。
      代替表面染色步骤,包括表面(抗小鼠CD3-eFluor 450,抗小鼠CD4-Alexa Fluor 700)和细胞内(抗小鼠IL-17A-PE和抗小鼠IL-22-APC)的所有抗体在固定和Perm/Wash后在一个单一的步骤中一起使用。我们已经用细胞内细胞因子以及针对CD3和CD4的表面抗体染色细胞
    9. 用200μl1x BD Perm/Wash TM缓冲液(300×g,5分钟,4℃)洗涤细胞两次。重悬在200μlFACS缓冲液中的沉淀。
    10. 通过FACS确定细胞内IL-17A和IL-22的水平来确认Th17细胞的谱系。良好的Th17分化将导致20-40%产生IL-17A的CD4T细胞。

数据分析

可以使用FACS Diva或Flow Jo软件分析数据。绘制线性FSC对SSC点图,并创建一个门(P1)以选择所有单元格。使用P1群体绘制线性CD3对CD4点图。大多数分化细胞对CD3和CD4都是阳性的。将分析CD3阳性群体(门P2)的细胞内IL-17和IL-22染色。

食谱

  1. 完全IMDM培养基(使用前在37°C预热)
    IMDM培养基+谷氨酰胺
    10%FBS
    1%青霉素/链霉素溶液
  2. 2x Th17分化条件培养基
    完全IMDM培养基(37℃预热),含有:
    10 ng/ml TGF-β1
    80ng/ml重组IL-6 20ng/ml重组IL-23 10μg/ml抗IFNγ 10μg/ml抗IL-4抗体
  3. FACS缓冲区
    PBS用
    0.5%牛血清白蛋白(BSA)
    0.05%叠氮化钠

致谢

作者要感谢以下PHS拨款的支持:P50HL084932,5R01HL061271和R37HL079142到J.K.K. P.K得到匹兹堡儿童医院研究咨询委员会从匹兹堡儿童医院获得的UPMC健康体系的支持。

参考文献

  1. Kumar,P.,Monin,L.,Castillo,P.,Elsegeiny,W.,Horne,W.,Eddens,T.,Vikram,A.,Good,M.,Schoenborn,AA,Bibby,K.,Montelaro ,RC,Metzger,DW,Gulati,AS和Kolls,JK(2016)。  肠道白介素-17受体信号介导肠道微生物群和自身免疫炎症的相互控制。 44(3):659-671。
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引用:Kumar, P. and Kolls, J. K. (2016). Lymphocyte Isolation, Th17 Cell Differentiation, Activation, and Staining. Bio-protocol 6(23): e2047. DOI: 10.21769/BioProtoc.2047.
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