Intracellular Staining for Phosphorylated STAT4 and STAT5 in Mouse Splenocytes
小鼠脾脏细胞中磷酸化STAT4和STAT5的胞内染色
发布: 2014年02月05日第4卷第3期 DOI: 10.21769/BioProtoc.1043 浏览次数: 13070
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Apr 2013
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Abstract
The Stat (Signal Transducer and Activator of Transcription) family of proteins are critical signal transducers involved in fundamental cellular processes, including cell growth and differentiation, development, apoptosis, immune responses and inflammation. In here, we describe a simple and reproducible flow cytometry protocol to measure Stat protein phosphorylation in splenocyte preparations from malaria infected mice.
Materials and Reagents
- C57BL/6 mice
- RBC lysing buffer (BD Biosciences, catalog number: 555899 )
- FACS buffer: Hanks balance salt solution (HBSS) with 2% foetal calf serum (FCS)
- Trypan blue (Sigma-Aldrich, catalog number: T8154 )
- AIM V® Medium (Life Technologies, catalog number: 31035025 )
- Recombinant IL-2 (eBioscience) (stock solutions prepared as recommended by manufacturer)br />
- Recombinant IL-12 (R&D Systems) (stock solutions prepared as recommended by manufacturer)
- 4% paraformaldehyde
- 90% ice-cold methanol
- CD4 antibody (GK1.5) (eBioscience)
- CD44 antibody (IM7) (eBioscience)
- CD62L antibody (MEL-14) (eBioscience)
- T-bet (4B10) antibody (eBioscience)
- Phosphorylated STAT4 antibody (at residue Y693, clone 38) (BD Biosciences)
- Phosphorylated STAT5 antibody (at residue Y694, clone 47) (BD Biosciences)
Equipment
- 6-well plates
- 70 μm cell strainer (BD Biosciences)
- Haemocytometer
- Refrigerated table top centrifuge
- LSR II (BD Systems)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Villegas-Mendez, A., de Souza, J. B., Lavelle, S., Findlay, E. G., Shaw, T. N., Saris, C. J., Hunter, C. A., Riley, E. M. and Couper, K. N. (2014). Intracellular Staining for Phosphorylated STAT4 and STAT5 in Mouse Splenocytes. Bio-protocol 4(3): e1043. DOI: 10.21769/BioProtoc.1043.
- Villegas-Mendez, A., de Souza, J. B., Lavelle, S. W., Gwyer Findlay, E., Shaw, T. N., van Rooijen, N., Saris, C. J., Hunter, C. A., Riley, E. M. and Couper, K. N. (2013). IL-27 receptor signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during malaria infection by repressing IL-12 dependent signals. PLoS Pathog 9(4): e1003293.
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分类
免疫学 > 免疫细胞染色 > 流式细胞术
生物化学 > 蛋白质 > 免疫检测 > 免疫染色法
生物化学 > 蛋白质 > 修饰
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