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        【求助】用流式细胞仪检测JAK-STAT5通路?

        丁香园论坛

        2153
        有谁用流式细胞仪检测过STAT-5磷酸化程度呀,求教具体方法,结果怎么样呀?
        1、制备样品细胞的单细胞悬液。
        2、多聚甲醛固定细胞。
        3、打孔剂在细胞膜上打孔。
        4、标记荧光素偶联的抗磷酸化STAT-5抗体。
        5、流式上样分析。

        Crucial role of interleukin-7 in T helper type 17 survival and expansion in autoimmune disease. Nature Medicine. 2010. 16(2):191-197
        这篇文章有用到该法。文章可以到《免疫学与病原生物学讨论版》置顶贴中找。
        kern6549 wrote:
        1、制备样品细胞的单细胞悬液。
        2、多聚甲醛固定细胞。
        3、打孔剂在细胞膜上打孔。
        4、标记荧光素偶联的抗磷酸化STAT-5抗体。
        5、流式上样分析。

        Crucial role of interleukin-7 in T helper type 17 survival and expansion in autoimmune disease. Nature Medicine. 2010. 16(2):191-197
        这篇文章有用到该法。文章可以到《免疫学与病原生物学讨论版》置顶贴中找。

        这篇文章中的方法写得非常简单,能否介绍1篇步骤稍稍详细的文章或把以上步骤描述得详细一点,谢谢!
        我上传几篇流式细胞仪检测蛋白磷酸化的详细说明,供大家实验参考
        题目:Single-cell phospho-protein analysis by flow cytometry
        再加一篇实战的
        题目:Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase.pdf
        题目:Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking
        题目:Intracellular Phospho-Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot
        题目:Intracellular phospho-protein staining techniques for flow cytometry Monitoring single cell signaling events
        题目:Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry
        题目:Flow cytometric detection of total and serine 473 phosphorylated Akt
        题目:Intracellular detection of Bcl-2 and p53 proteins by flow cytometry comparison of monoclonal antibodies and sample preparation protocols
        Intracellular phospho‐protein staining techniques for flow cytometry Monitoring single cell signaling events
        Measurement of MAP kinase Activation by Cytometry Using Phospho-Specific Antibodies to MEK
        Multiparametric flow cytometric analysis in a breast cancer cell line (MCF-7)
        Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry

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