人Th亚群分化方案

人Th亚群分化方案

准备培养基
1640培养基、血清(胎牛)、双抗、L-谷氨酰胺、B-巯基乙醇

培养基配置
RPM-1640+10%胎牛血清+1mML-谷氨酰胺+55μM B-基乙醇+双抗

准备细胞
分选出Naive CD4*T cells(CD4*CD25-CD44!0CD62Lhigh)

细胞刺激培养
1.CD3包板:包板浓度2μg/ml(96孔板加50ul,48孔板加200ul)加入上述体积后4度过夜包被,包被后弃去孔内液体，PBS洗板2次。
2.分选的Naive CD4*Tcells加入包被孔内。
 a)96孔板加入1*105细胞(200y培养体系)
 b)48孔板加入2*105细胞(500μl培养体系)
3.配置分化培养基(4μl/mlanti-human CD28+10 ng/ml HumanIL2)
根据需要分化的方向额外添加以下成分:
a)TH1,IL-12 (50 ng/ml) plus anti-human lL-4 (10 ug/ml)
b)TH2,IL-4(50ng/ml) plus anti-human lFN-y(10 ug/ml)
c)TH17,1L-6 (50 ng/ml),iL-1β (10 ng/ml),lL-23 (10 ng/ml), TGF-β (10 ng/ml)plus-an-ti-human lFN-y(10 ug/ml) and anti-human lL-4 (10 ug/ml)
e) TH9,IL-4 (50 ng/ml),TGF-β (10 ng/ml), plus anti-human lFN-y(10ug/ml)
f)TH22,1L-6 (50 ng/ml),L-23(50 ng/ml), plus anti-human lFN-y (10 ug/ml) andanti-human lL-4 (10 ug/ml)
g)Tfh,1L-6(50 ng/ml),IL-21 (10 ng/ml),cxCL13 (10 ng/ml).

4.细胞培养分化3-5天,培养过程中若培养液上清变黄,更换培养基,一般建议半量换液,根据培养经验，一般是隔天换液。
5.培养分化后鉴定(胞内染色):分化后的细胞加入20ng/mPMA,lug/mllonomycin刺激30min后,加入10ug/mIBFA阻断,继续培养3.5h后收集细胞进行流式检测。
6.细胞鉴定:购买相应的流式抗体及辅助试剂进行胞内染色鉴定。
 

细胞鉴定所需试剂:

参考文献:
1.Carbo A, Hontecillas R, Andrew T, et al. Computational modeling of heterogeneity andfunction of CD4*Tcells. Front Cell Dev Biol. 2014;2:31.
2.O'Conor W, Jr., Zenewicz LA, Flavell RA. The dual nature of T(H)17 cells: shifting thefocus to function. Nat lmmunol. 2010;11:471-476.
3.Schmitt N, Ueno H. Regulation of human helper T cell subset differentiation by cytokines. Curr Opin lmmunol. 2015;34:130-136.
4.Crotty S. T follicular helper cell differentiation, function, and roles in disease. lmmunity.2014:41:529-542.
5.Gegnat J, Paroni M, Maglie S, et al. Plasticity of human CD4 Tcell subsets. Front lmmu-nol. 2014:5:630.