流式检测CD4+ T细胞亚型转录因子   

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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,4 °C 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也是有效的。


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

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  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.
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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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