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胞内染色法检测T细胞活化产生的细胞因子   

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摘要:T细胞经过抗原刺激活化后,会发生一系列的变化 (Yang等,2014a和2014b; Pu等,2016),如表达表面活性分子、产生特定的细胞因子、细胞增殖分裂及细胞凋亡等,利用流式细胞仪可以在单细胞水平上对其进行多参数检测 (Yang等,2014b),如:活化T细胞的表面分子 (如CD44、CD69等) 及其相应的效应分子 (如Granzyme B、IFN-γ及Perforin等),从而对抗原特异性免疫反应在细胞水平进行定性和定量分析。本次实验中,我们利用含OVA模式抗原的小鼠黑色素瘤细胞B16-OVA对OT-IT细胞给予刺激,24h后检测胞内细胞因子IFN-γ的表达,对照组OT-IT细胞无肿瘤细胞刺激。

关键词: 流式检测, T细胞活化, 细胞因子, 胞内染色

材料与试剂

  1. 96孔板 (Jet Biofil, catalog number: TCP001096)
  2. OT-I小鼠 (Jackson Lab)
  3. Anti-mCD8-AF700 (BioLegend, catalog number: 100730)
  4. Anti-IFN-γ-APC (BioLegend, catalog number: 505810)
  5. anti-CD16/32 抗体 (clone 2.4G2) (BioLegend, catalog number: 101301)
  6. B16-OVA细胞 (芝加哥大学Hans Schreiber馈赠)
  7. FBS (Gibco, catalog number: 10270-106)
  8. PBS (Hyclone, catalog number: SH30256.01B)
  9. RPMI 1640培养基 (Hyclone, catalog number: SH30027.01)
  10. 双抗Penicillin/Streptomycin (Hyclone, catalog number: SV30010)
  11. 10x Permeabilization Buffer (eBioscience, catalog number: 00-8333-56)
  12. BFA (Brefeldin A) (BioLegend, catalog number: 420601)
  13. 多聚甲醛 (Adamas, catalog number: 46556A)
  14. NaCl (Sigma-Aldrich, catalog number:V900058-500g)
  15. KCl (Sigma-Aldrich, catalog number:V900068-500g)
  16. Na2HPO4 (Sigma-Aldrich, catalog number:V900060-500g)
  17. KH2PO4 (Sigma-Aldrich, catalog number:V900041-500g)
  18. NaN3 (Amresco, catalog number: 0639-250g)
  19. NaOH (Sigma-Aldrich, catalog number: V900797)
  20. 10× PBS (1 L, pH = 7.3) (见溶液配方)
  21. FACS buffer (见溶液配方)
  22. 4%多聚甲醛 (见溶液配方)

仪器设备

  1. 生物安全柜 (LabGard, model: Class II/Labconco) 
  2. 离心机 (Eppendorf, model: 5810R) 
  3. 光学显微镜 (Olympus, model: IX2-SLP)
  4. 流式细胞仪 (Backman Coulter, model: CytoFLEX S)
  5. 移液器 (Eppendorf)
  6. 二氧化碳培养箱 (Panasonic, model: MCO-20AIC)
  7. 高压蒸汽灭菌锅 (Boxun,model: YXQ-LS-100SII)

软件

  1. FlowJo 软件

实验步骤

  1. 获取OT-I小鼠的脾脏细胞,计数,按照1 × 105~2 × 105接种于96孔板,培养基为含IL-2的RPMI完全培养基,按照 (OT-I小鼠的脾脏细胞:B16-OVA肿瘤细胞) E:T = 3:1的比例加入B16-OVA肿瘤细胞刺激,对照组不加肿瘤细胞;
  2. 刺激后约20 h,加入BFA反应2.5 h~4 h,用排枪吸取悬浮细胞悬液加入新的96孔板 (U型),500 x g离心3 min,弃去上清;
  3. 加入FACS buffer,200 μl/sample,500 x g离心3 min,弃去上清;
  4. 细胞表面分子染色,单份样品抗体用量如下,按照50 μl/sample加入样品,适度混匀,4 °C孵育25 min:
    25 μl FACS buffer
    25 μl 2.4G2 (200 ng/ml)
    0.15 μl anti-mCD8-AF700;
  5. 加入FACS buffer 150 μl/sample, 500 x g离心3 min,弃去上清;
  6. 重复第5步;
  7. 细胞固定,按照100 μl/sample标准加入4% PFA固定,加入后立即使用移液器反复吹打混匀防止细胞结块,室温孵育15 min;
  8. 加入FACS buffer 150 μl/sample,500 x g离心3 min,弃去上清;
  9. 重复第8步;
  10. 破膜处理,新鲜配制1× Permeabilization Buffer,按照200 μl/sample加入1× Permeabilization Buffer,用移液器吹打重悬细胞,室温15 min,破膜完成后,500 x g,离心3 min,去上清;
  11. 胞内细胞因子染色,单份样品抗体用量如下,并按照50 μl/sample加入样品,适度混匀,4 °C孵育25 min:
    50 μl 1x Permeabilization Buffer
    0.15 μl anti-IFN-γ-APC;
  12. 加入1× Permeabilization Buffer 150 μl/sample,500 x g离心3 min,弃去上清;
  13. 用FACS buffer 重悬样品 (70 μl/sample),将样品置于冰上;
  14. 使用流式细胞仪分析获取数据,利用FlowJo 软件分析流式检测结果。

结果与分析

胞内染色法检测T细胞活化引起的IFN-γ的表达情况

结果分析:图1A通过FSC-A和SSC-A分析排除死细胞和细胞碎片,确定和收集主要细胞群;图1B进一步通过APC-A700-A染色确定CD8+ T细胞;图1C~1D在图1B基础上通过APC-A染色确定IFN-γ的表达。


图1. 胞内染色检测活化T细胞释放IFN-γ的情况

注意事项

  1. 该实验中的200 ng/ml为2.4G2抗体最终浓度;抗体Anti-mCD8-AF700及Anti-IFN-γ- APC具有光谱重叠需要预先在流式细胞仪上调好补偿,多色共染时对于抗体的选择需要注意,要尽量减少光谱重叠,例如两色共染时可选择APC + FITC标记或者APC + AF488标记,这样可以有效避免光谱重叠。
  2. 本实验中也可以收集24 h时细胞上清,利用ELISA或CBA的方法检测上清中分泌IFN-γ的表达,胞内染色的优点在于能够明确产生IFN-γ的细胞类型,此外,其他效应分子如Granzyme B、Perforin及TNF-α等也可以用同样的胞内染色方法进行流式检测。

溶液配方

  1. 10× PBS (1 L, pH=7.3)
    NaCl
    80 g
    KCl
    10 g
    Na2HPO4
    14.4 g
    KH2PO4
    2.4 g
    将上述试剂加入800 ml ddH2O依次进行溶解,待所有试剂完全溶解后,高温湿热灭菌,冷却后室温保存待用
  2. FACS buffer
    10× PBS
    100 ml
    FBS
    10 ml
    20% NaN3
    2.5 ml
    使用量筒取100 ml 10× PBS,首先加入800 ml ddH2O进行稀释,再加入10 ml FBS和2.5 ml 20% NaN3,混合均匀后,加入ddH2O定容至1 L,4 °C备用
  3. 4%多聚甲醛
    多聚甲醛
    4.0 g
    PBS
    100 ml
    称量4.0 g多聚甲醛,先加入80 ml已配制好的1× PBS在60~65 °C加热搅拌溶解,20分钟后,使用NaOH调节pH至7.0,待溶液呈现清亮,无未溶解固体成分,加入PBS定容至100 ml,避光保存于4 °C待用
    注:甲醛具有较强的毒性,注意做好防护措施。

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。本实验方案用于已发表的文章Han 等,2019;Qi等,2019;Zhang等2019。

参考文献

  1. Han, P., Dai, Q., Fan, L., Lin, H., Zhang, X., Li, F. and Yang, X. (2019). Genome-wide CRISPR screening identifies JAK1 deficiency as a mechanism of T-Cell resistance. Front Immunol 10: 251.
  2. Pu, Y., Xu, M., Liang, Y., Yang, K., Guo, Y., Yang, X. and Fu, Y. X. (2016). Androgen receptor antagonists compromise T cell response against prostate cancer leading to early tumor relapse. Sci Transl Med 8(333): 333ra347.
  3. Qi, X., Li, F., Wu, Y., Cheng, C., Han, P., Wang, J. and Yang, X. (2019). Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcgammaR affinity. Nat Commun 10(1): 2141.
  4. Yang, X., Zhang, X., Fu, M. L., Weichselbaum, R. R., Gajewski, T. F., Guo, Y. and Fu, Y. X. (2014a). Targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses. Cancer Cell 25(1): 37-48.
  5. Yang, X., Zhang, X., Sun, Y., Tu, T., Fu, M. L., Miller, M. and Fu, Y. X. (2014b). A BTLA-mediated bait and switch strategy permits Listeria expansion in CD8α(+) DCs to promote long-term T cell responses. Cell Host Microbe 16(1): 68-80.
  6. Zhang, X., Cheng, C., Hou, J., Qi, X., Wang, X., Han, P. and Yang, X. (2019). Distinct contribution of PD-L1 suppression by spatial expression of PD-L1 on tumor and non-tumor cells. Cell Mol Immunol 16(4): 392-400.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:韩萍, 杨选明. (2019). 胞内染色法检测T细胞活化产生的细胞因子. Bio-101: e1010314. DOI: 10.21769/BioProtoc.1010314.
How to cite: Han, P. and Yang, X. M. (2019). Analyzing Cytokines Production during T Cell Activation by Flow Cytometry Intracellular Staining. Bio-101: e1010314. DOI: 10.21769/BioProtoc.1010314.
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