Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells

Fredine T. Lauer, Jesse L. Denson, Scott W. Burchiel
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引用次数: 17

Abstract

This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and phenotype stability are not altered through cryopreservation and extended storage. For this analysis, HPBMC were isolated from 7 healthy individuals, and 11-color flow cytometry was performed on freshly isolated samples as well as samples cryopreserved for short- and long-term periods. There is no significant difference in the percentage of cells expressing the CSM CD3, CD4, CD8, CD45RO, CD16, CD19, or CD56 between freshly isolated and cryopreserved HPBMC. Hence, cryopreservation of HPBMC does not influence the phenotype of distinct cellular subsets in isolated mononuclear cells. This protocol for HPBMC isolation, cryopreservation, and thawing of HPBMC is intended for long-term studies of large cohorts requiring sample shipment and subsequent batch analysis. © 2017 by John Wiley & Sons, Inc.

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人外周血单个核细胞的分离、冷冻保存和免疫表型分析
本单元描述了人类外周血单个核细胞(hbmc)的分离、冷冻保存和解冻的程序,以及使用多色流式细胞术分析用于免疫表型的细胞表面标记物(CSM)。这种方法可以用来确保细胞的完整性和表型稳定性不改变,通过低温保存和延长储存。为了进行这项分析,从7名健康个体中分离出HPBMC,并对新鲜分离的样本以及短期和长期冷冻保存的样本进行11色流式细胞术。在新鲜分离和冷冻保存的HPBMC中,表达CSM CD3、CD4、CD8、CD45RO、CD16、CD19或CD56的细胞百分比没有显著差异。因此,HPBMC的低温保存不会影响分离的单核细胞中不同细胞亚群的表型。本HPBMC分离、低温保存和解冻方案适用于需要样品运输和后续批量分析的大型队列的长期研究。©2017 by John Wiley &儿子,Inc。
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