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标题：Optimized flow cytometric protocol for the detection of functional subsets of low frequency antigen-specific CD4 + and CD8 + T cells
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作者：Marie Mura ; Sidhartha Chaudhury ; Fouzia Farooq 等
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2020
卷号：7
页码：1-12
DOI：10.1016/j.mex.2020.101005
语种：English
出版社：Elsevier
摘要：AbstractDetection of low-frequency cells using flow cytometry is challenging, as the sensitivity of the analysis is dependent on the signal-to-noise ratio, and a cell frequency of 1 in 10,000 cells is accepted as the lower limit of detection for standard flow cytometry. A solution to this problem is to pre-enrich rare cell populations using magnetic-bead conjugated antibodies targeting lineage or activation markers. For measuring vaccine or pathogen induced immune responses, this method drastically increases the signal-to-noise ratio by enriching only activated (i.e., antigen-specific) cells and excluding all other peripheral blood leukocytes from the subsequent analysis. To date, magnetic enrichment of antigen-specific cells has only been described for CD4+T cells processed for surface staining. The current study significantly expands the methodology to allow detection of antigen-specific CD8+T cells and analysis of cells that had been processed for intracellular staining.•The protocol described here allows magnetic enrichment of PBMCs after fixation and intracellular staining steps without increasing the non-specific background.•The protocol is adapted to automated enrichment-mode on flow cytometers.•The procedure boosts the sensitivity of the flow cytometry analysis by significantly increasing the sample size of functional antigen-specific cells without skewing the composition of the functional cells pool.Graphical abstractDisplay Omitted
关键词：Flow cytometry;Intracellular staining;Cell enrichment;Antigen-specific cell detection;Rare cell detection;Cytokine response