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

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. MojoSort^TM 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