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Analyzing Transcription Factors of CD4+ T Cell Subtypes by Flow Cytometry   

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摘要:Naïve CD4+ T细胞在不同的条件下可诱导形成不同的CD4+ T细胞亚型,比如,Th1 (Szabo等,2002),Th2 (Mowen和Glimcher,2004;Flaherty和Reynolds,2015),Treg (Sekiya和Yoshimura,2016;Read等,2019),Th9 (Kaplan等2015),Th17 (Chung等,2009;Rumble和Segal, 2014)。不同CD4+ T细胞亚型表达特异的转录因子,具有不同的免疫学功能,特异转录因子表达是鉴定不同CD4+ T细胞亚群的关键指标。本实验以检测转录因子FoxP3+的CD4+ T细胞分析调节性T细胞Treg细胞比例为例,演示体外分化后CD4+ T细胞亚型转录因子的流式检测。

关键词: T 细胞分化, CD4+ T细胞亚型, Cytokines, 转录因子

材料与试剂

  1. 96孔板U型板 (Jet Biofil, catalog number: TCP001096)
  2. Anti-mCD4 FITC抗体 (BioLegend, catalog number: 100405)
  3. Anti-FoxP3 抗体 (Thermo Fisher, eBioscience, catalog number: MA5-16224)
  4. 2.4G2 (anti-CD16/32 抗体) (BioLegend, catalog number: 101301)
  5. 阻断抗体和细胞因子
    anti-mouse CD3
    BioLegend
    100302
    克隆号 145-2C11;
    anti-mouse CD28
    BioLegend
    102102
    克隆号 37.51;
    anti-mouse IL4
    BioLegend
    504102
    克隆号 11B11;
    anti-mouse IL12
    义翘神州
    CT022-RP01;

    anti-mouse IL2
    BioLegend
    503702
    克隆号 JES6-1A12;
    anti-mouse IFNγ
    BioLegend
    505802
    克隆号 XMG1.2;
    mouse IL2
    BioLegend
    575406;

    mouse IFNγ
    BioLegend
    751806;

    mouse IL12
    BioLegend
    577006;

    mouse IL4
    BioLegend
    574306;

    mouse IL6
    BioLegend
    575706;

    mouse IL1β
    义翘神州
    50101-MNAE;

    mouse TGF-β1
    BioLegend
    763104;

    anti-Hamster Ig (H + L) Jackson
    ImmunoResearch
    127005160

  6. FBS (Gibco,catalog number: 10270-106,55 °C灭活0.5 h)
  7. PBS (Hyclone, catalog number: SH30256.01B)
  8. RPMI-1640培养基 (Hyclone, catalog number: SH30027.01)
  9. Penicillin/Streptomycin (Hyclone, catalog number: SV30010)
  10. Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher, eBioscience, catalog number: 00-5523-00)
  11. MojoSortTM Mouse CD4 Naïve T Cell Isolation Kit (BioLegend, catalog number: 480040)
  12. Foxp3 Fixation/Permeabilization buffer
  13. NaCl
  14. KCl
  15. Na2HPO4
  16. KH2PO4
  17. Milli-Q H2O
  18. β-巯基乙醇
  19. L-Glutamine
  20. 10× PBS buffer (见溶液配方)
  21. 1× PBS buffer (见溶液配方)
  22. FACS buffer (见溶液配方)
  23. RPMI-1640完全培养基 (见溶液配方)
  24. 2.4G2 buffer (见溶液配方)

仪器设备

  1. 流式细胞仪 (Cytoflex S)
  2. 电动吸引器 (斯曼峰,model: YX930D)
  3. 移液器 (Eppendorf)
  4. 正置光学显微镜 (Olympus, model: IX2-SLP)
  5. 离心机 (Eppendorf, model: 5810R)
  6. 医用低温保存箱 (海尔,model: DW-25L262/BD-226W)
  7. 振荡混匀机 (Kylin-bell)
  8. 生物安全柜 (LabGard, model: Class II/Labconco)
  9. 二氧化碳培养箱 (Panasonic, model: MCO-20AIC)

软件

  1. FlowJo

实验步骤

用eBioscienceTM FoxP3/Transcription Factor Staining Buffer Set试剂盒进行CD4+ T细胞亚型转录因子的流式染色。染色体系为96孔U型板。

  1. 抗体包被:1× PBS稀释抗体anti-hamster Ig (H + L) (2 µg/ml)。将稀释抗体100 µl/well加入96孔板,4 °C过夜;
  2. 取小鼠脾脏,纯化naïve CD4T细胞,将纯化的CD4T细胞种在包被过夜的96孔板,加anti-mCD3 (0.25 µg/ml)和anti-mCD28 (1 µg/ml)刺激,根据CD4+ T细胞的体外分化条件诱导成不同亚型的CD4+T细胞。诱导不同CD4+ T细胞亚型需要的阻断抗体和细胞因子如下:
    Th1: 10 μg/ml anti-IL-4,5 ng/ml IL-12,250 U/ml IL-2
    Th2: 5µg/ml anti-IFN-γ,10 ng/ml IL-4,250 U/ml IL-2
    Th9: 10 μg/ml anti-IFN-γ,10 ng/ml IL-4,2 ng/ml TGF-β1
    Th17: 10 μg/ml anti-IL-4,10 μg/ml anti-IFN-γ,50 ng/ml IL-6,2 ng/ml TGF-β1
    Treg: 1 μg/ml anti-IFN-γ, 1 μg/ml anti-IL-4,2ng/ml TGF-β1
    用RPMI-1640完全培养基培养3~4 d;
  3. 收集细胞:取 105细胞置于96孔U型板,加入100 µl的FACS buffer,500 x g离心5 min,弃去上清;
  4. 表面检测抗体配制:将 0.15 µl anti-mouse CD4 FITC抗体加入到25 µl FACS buffer和25 µl 2.4G2 (200 ng/ml)的混合液中,混匀 (根据样品数量配制适当体积的抗体); 
  5. 表面抗体染色:每个样品加入50 µl配好的抗体,Vortex混匀,4 °C 避光染色25 min;
  6. 准备固定破膜液和破膜液:固定破膜液按照需要配制合适体积的Foxp3 Fixation/Permeabilization buffer,按要求将稀释液和浓缩液3:1混合,破膜液用ddH2O将10× Permeabilization Buffer稀释成1× Permeabilization Buffer;
  7. 洗去残余抗体:将步骤4中染色完成的样品加入150 µl FACS buffer,500 x g离心5 min,弃去上清,重复该步骤;
  8. 固定破膜:加入步骤5中配制的固定破膜液FoxP3 Fixation/Permeabilization Buffer 100 µl, vortex混匀,常温避光30~60 min;
  9. 洗去固定液:加入100 µl步骤5中配好的破膜液1× Permeabilization Buffer,500 x g离心5 min,弃去上清,重复该步骤;
  10. 转录因子染色抗体配制和染色:0.15 µl转录因子抗体 + 50 µl 1× Permeabilization Buffer,CD4T细胞亚型对应的转录因子如下:
    Th1: TBX21
    Th2: GATA3
    Th9: PU.1 和IRF4
    Th17: ROR-gt
    Treg: FoxP3
    加入配制的转录因子抗体到样品,Vortex 混匀,4 °C 避光染色25 min;
  11. 洗去残余转录因子抗体:加入150 µl 1× Permeabilization Buffer,500 x g离心5 min,弃去上清,重复该步骤;
  12. 重悬流式分析:用60 µl的FACS buffer重悬样品,流式细胞仪分析样品及FlowJo分析FACS结果。

结果与分析

以Treg细胞的转录因子Foxp3为例,展示转录因子的流式染色 (图1)。
结果分析:纯化的naïve CD4T细胞是未分化的T细胞,在诱导形成FoxP3+的Treg细胞前是很少表达FoxP3的,诱导前的流式细胞染色仅有4% 的细胞是FoxP3+的。在向Treg细胞诱导4 d后。CD4+T细胞中有37% 的细胞表达FoxP3。该结果不仅说明该条件是可以定向诱导naïve CD4+ T细胞分化形成Treg细胞,也说明该检测转录因子的方法是有效的。


图1. 流式检测诱导前后Treg细胞的比例

失败经验

  1. 为防止染色不均匀,染色前后都要震荡混匀;为防止液体溅出,调整vortex至合适的转速。
  2. 每次流式仅取少量的细胞,以防止一次实验意外导致无细胞可重复。T细胞体外分化也要多做一个重复,每个重复仅取部分细胞混匀后鉴定。

溶液配方

  1. 10× PBS buffer
    NaCl             80 g
    KCl                10 g
    Na2HPO4     14.4 g
    KH2PO4        2.4 g
    加入Milli-Q H2O 去离子水定容1 L
  2. 1× PBS buffer
    将10× PBS 100ml加入Milli-Q H2O 去离子水定容1 L
    pH 7.2~7.4,高压灭菌
  3. FACS buffer
    1× PBS buffer    1 L
    血清                     20 ml
    NaN3                   0.05%
  4. RPMI-1640完全培养基
    RPMI-1640培养基
    9% 胎牛血清
    1% Penicillin-Streptomycin
    55 μM β-巯基乙醇
    L-Glutamine 200mM
  5. 2.4G2 buffer
    10 µg 2.4G2加入50 ml FACS buffer 配成200 ng/ml

致谢

感谢杨选明实验室全体成员的建议和帮助。该研究受国家自然基金 (81671643)、科技部重点研发计划 (2016YFC1303400) 资助。

参考文献

  1. Chung, Y., Chang, S. H., Martinez, G. J., Yang, X. O., Nurieva, R., Kang, H. S., Ma, L., Watowich, S. S., Jetten, A. M., Tian, Q. and Dong, C. (2009). Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 30(4): 576-587.
  2. Flaherty, S. and Reynolds, J. M. (2015). Mouse Naive CD4+ T cell isolation and in vitro differentiation into T cell subsets. J Vis Exp 98. 
  3. Kaplan, M. H., Hufford, M. M. and Olson, M. R. (2015). The development and in vivo function of T helper 9 cells. Nat Rev Immunol 15(5): 295-307.
  4. Mowen, K. A. and Glimcher, L. H. (2004). Signaling pathways in Th2 development. Immunol Rev 202: 203-222.
  5. Read, K. A., Powell, M. D., Sreekumar, B. K. and Oestreich, K. J. (2019). In vitro differentiation of effector CD4(+) T Helper cell subsets. Methods Mol Biol 1960: 75-84.
  6. Rumble, J. and Segal, B. M. (2014). In vitro polarization of T-helper cells. Methods Mol Biol 1193: 105-113.
  7. Sekiya, T. and Yoshimura, A. (2016). In vitro Th differentiation protocol. Methods Mol Biol 1344: 183-191.
  8. Szabo, S. J., Sullivan, B. M., Stemmann, C., Satoskar, A. R., Sleckman, B. P. and Glimcher, L. H. (2002). Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295(5553): 338-342.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:李民, 杨选明. (2019). 流式检测CD4+ T细胞亚型转录因子. Bio-101: e1010315. DOI: 10.21769/BioProtoc.1010315.
How to cite: Li, M. and Yang, X. M. (2019). Analyzing Transcription Factors of CD4+ T Cell Subtypes by Flow Cytometry. Bio-101: e1010315. DOI: 10.21769/BioProtoc.1010315.
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