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Date:
2020-09-05
[Abstract] In this report, we present the implementation of mass cytometry for intracellular staining using fixed whole blood. In our assay described here, 250 µl of whole blood, is stimulated in vitro with PMA/ionomycin (or left unstimulated), in the presence of secretion inhibitors (brefeldin A and monensin), lysed-fixed using SMART TUBE buffers, barcoded (optional), surface stained, fixed, stained for intracellular markers, fixed and DNA stained. Using 250 µl of whole blood from a healthy donor, we show that the expression of major lineage populations such as T cells, B cells, NK cells and monocytes, as well as cytokines such as CD4+ and CD8+ IFNγ and TNFα across multiple batches (n = 27) is consistent, with the co-efficient of variation (CVs) ≤ 21%, implying ...
[摘要] [摘要] 在本报告中,我们介绍了使用固定全血在细胞内染色中进行细胞计数的方法。在此处所述的测定中,在存在分泌抑制剂(布雷菲德菌素A和莫能菌素)的情况下,用PMA /离子霉素体外刺激(或不刺激)250 µl全血,使用SMART TUBE缓冲液裂解固定,加条形码(可选) ),表面染色,固定,细胞内标记物染色,固定和DNA染色。使用250微升的全血从健康供体中,我们表明,主要的谱系种群,例如T细胞,B细胞,NK细胞和单核细胞,以及细胞因子,例如CD4的表达+ 和CD8 + IFN γ 和TNF α 多个批次(n = 27)之间的一致性是一致的,变异系数(CV)≤21 %,这意味着最小的变异性。对于每种主要细胞类型,该百分比报告为单峰百分比。据报道,响应刺激的细胞因子表达的百分比为直接亲代细胞类型的百分比。该方案具有许多好处:从生物学的角度来看,它可以应用于临床研究,尤其是在抽血量有限的情况下。从技术上讲,该协议可适用于条形码,这增加了更均匀的样品染色以及抗体保守性的好处,特别是对于大型研究人群。最后,对于包括当前全球COVID-19大流行在内的涉及传染病的研究,该方案允许在处理和染色之前固定传染性样品,从而降低了生物安全风险。
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