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BioGems
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PEPROTECH
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GIBCO
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已认证品牌介绍
BioGems
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PEPROTECH
试剂
已认证品牌介绍
GIBCO
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小鼠Th亚群分化方案
142 人阅读发布时间：2025-09-18 09:23
小鼠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、β -巯基乙醇
培养基配置
RPMI-1640+10%胎牛血清+1mML-谷氨酰胺+55μM B-巯基乙醇+双抗
准备细胞
分选出Naive CD4*Tcells(CD4*CD25-CD440CD62Lhigh)
细胞刺激培养
1.CD3包板:包板浓度2μg/ml(96孔板加50ml,48孔板加200ml)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(200ml培养体系b)48孔板加入2*105细胞(500ul培养体系)
3.配置分化培养基(1μg/mlanti-mouse CD28)
按需要分化的方向加入以下分化条件的培养基:
a)Th0: no cytokines/antibodies +20 ng/ml lL-2;
b)Th1:20 ng/mllL-12+20 ng/mllL-2;plus 10ug/mL anti-mouse lL-4;
c)Th2:10ng/mllL-4+20 ng/mllL-2;plus 10 ug/ml anti-mouse lFy;
d)Th17:5ng/mlTGF-β,20 ng/mllL-6,10ng/ml1L-23; plus 10ug/ml anti-mouseIFNy.10 ug/ml anti-mouse lL-4;
e)Tfh:20 ng/mllL-6,20 ng/lL-21; plus 10 ug/ml anti-mouse lFNy,10 ug/mlanti-mouselL-4&10ug/ml anti-mouse TGFβ;f)Treg: 20 ng/mlIL2+5ng/mlTGF-B.
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,1ug/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。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。
细胞分化所需细胞因子及抗体:
| 生产商 | 产品编号 | 产品名称 | 产品规格 |
| PeproTech | 212-12 | Recombnant Murne IL-2 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 214-14 | Recombinant Murine lL-4 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 216-16 | Recombinant Murine lL-6 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 210-12 | Recombinant Murine lL-12 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 210-21 | Recombinant Murine lL-21 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 210-23 | Recombinant Murine lL-23 | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 100-21 | Human TGF-beta1-Mammalian | 5 ug/20 ug/50 μg/100 ug/250 μg/ 500 ug/1000 μg |
| PeproTech | 05112-25 | Anti-Mouse CD3 SAFIRE Purified | 100 μg/500 μg |
| PeproTech | 10312-25 | Anti-Mouse CD28 SAFIRE Purified | 100 μg/500 μg |
| PeproTech | 80812-25 | Anti-Mouse lFN gamma SAFIRE Purified | 100 μg/500 μg |
| PeproTech | 81112-25 | Anti-Mouse IL-4 SAFIRE Purified | 100 μg/500 μg |
细胞鉴定所需试剂:
| 生产商 | 产品编号 | 产品名称 | 产品规格 |
| BioGems | 05111-40 | Anti-Mouse CD3 BG Violet 450 | 25 μg/100 μg |
| BioGems | 10111-50 | Anti-Mouse CD4 FITC | 25 μg/100 μg |
| BioGems | 80812-77 | Anti-Mouse lFN gamma PE-Cy7 | 25 μg/100 μg |
| BioGems | 81112-80 | Anti-Mouse IL-4 APC | 25 μg/100 μg |
| BioGems | 73812-60 | Anti-Mouse IL-17A PE | 50 μg |
| BioGems | 42813-77-100 | Rat lgG1 lsotype Control PE-Cy7 | 100 μg |
| BioGems | 42813-80-100 | Rat lgG1 lsotype Control APC | 100 μg |
| BioGems | 41813-60-100 | Rat lgG2a lsotype Control PE | 100 μg |
| BioGems | 2031560-5mg | Brefeldin A | 5mg |
| BioGems | 5608212-1mg | lonomycin (calcium salt) | 1mg |
| BioGems | 1652981-1mg | Phorbol 12-myristate 13-acetate | 1mg |
| BioGems | 92110-00-150 | Permeabilization Buffer (10X) | 150ml |
| BioGems | 92160-00-160 | Transcription Factor Fixation/Permeabilization Diluent | 160ml |
| BioGems | 92550-00-50 | Transcription Factor Fixation/Permeabilizatio Concentrate | 50ml |
参考文献:
1. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for the generation ofpathogenic effector TH17 and regulatory T cells. Nature.2006;441:235-238.
2. Nurieva Rl, Chung Y, Hwang D, et al. Generation of T follicular helper cells is mediatedby interleukin-21 but independent of T helper 1, 2,or 17 cell lineages. lmmunity.2008;29:138-149.
3. Zhu J, Paul WE. Peripheral CD4* T-cell differentiation regulated by networks of cytokines and transcription factors. lmmunol Rev. 2010;238:247-2624. Zhou L, Chong MM, Littman DR. Plasticity ofCD4* Tcell lineage differentiation. lmmu
nity.2009;30:646-655