流式细胞术检测表面标记的人外周血单个核T细胞的细胞内细胞因子。

Fredine T Lauer, Jesse L Denson, Ellen Beswick, Scott W Burchiel
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引用次数: 7

摘要

在本系列的最新单元中,介绍了使用细胞表面标记(CSM)染色和多色流式细胞术分析从外周血中分离的HPBMC的分离,冷冻保存,解冻和免疫表型分析的方案。目前的程序描述了CSM和细胞内标记物(ICM)的检测和定量,包括转录因子和细胞因子,在CD4+ t细胞激活和分化后使用多色流式细胞术。结果表明,可以在表面标记定义的T细胞中获得可重复和可靠的ICM检测,以识别细胞的功能亚群。在分析的8种表型(T-CD3、Th-CD4、Tmem-CD45RO、活化T-CD3/CD25、Treg- Foxp3/CD25、Th1-IFNγ、Th2- IL-4、Th17-IL-17A)中,新鲜和冷冻保存的HPBMC没有差异。与新鲜分离的样品相比,在短期(30-90天)冷冻保存的样品中观察到活化T- CD3/CD69的差异,这可能是由于对照差异或样本量小所致。©2017 by John Wiley & Sons, Inc。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

Intracellular Cytokine Detection by Flow Cytometry in Surface Marker-Defined Human Peripheral Blood Mononuclear T Cells.

In a recent unit in this series, protocols for the isolation, cryopreservation, thawing, and immunophenotyping of HPBMC isolated from peripheral whole blood using cell surface marker (CSM) staining and multi-color flow cytometry analysis were presented. The current procedure describes the detection and quantification of CSM and intracellular markers (ICM), including transcription factors and cytokines, following activation and differentiation of CD4+ T-cells using multi-color flow cytometry. Results indicated that repeatable and robust detection of ICM could be obtained in surface marker-defined T cells that identify functional subsets of cells. There were no observed differences between fresh and cryopreserved HPBMC in eight phenotypes analyzed (T-CD3, Th-CD4, Tmem-CD45RO, activated T-CD3/CD25, Treg- Foxp3/CD25, Th1-IFNγ, Th2- IL-4, Th17-IL-17A). There was an observed difference in activated T- CD3/CD69 in the short term (30-90 days) cryopreserved samples as compared to the freshly isolated samples, which may have resulted from the variance in controls or small sample size. © 2017 by John Wiley & Sons, Inc.

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