OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.
OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.
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OMIP-102：对 T 细胞和树突细胞进行深入评估，对人体免疫系统进行 50 色表型分析。
DOI：
10.1002/cyto.a.24841
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Mair,Florian
中科院分区：
文献类型：
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作者：
Konecny,AndrewJ;Mage,PeterL;Tyznik,AaronJ;Prlic,Martin;Mair,Florian
我们报告了一种优化的 50 色光谱流式细胞术面板的开发，该面板旨在深入分析人类血液和组织中的免疫系统，目的是最大限度地利用当前可用的流式细胞术平台收集的信息量。我们使用外周血单核细胞 (PBMC) 建立并测试了该组合，但包含了 CD45，以使其将来能够用于人体组织样本的分析。该面板包含所有主要免疫细胞亚群的谱系标记，以及一组广泛的表型标记，重点关注 T 细胞和树突状细胞 (DC) 区室的激活和分化状态。我们概述了从同时测量如此大量的蛋白质中可以获得的生物学见解，并提出这种方法为全面探索具有有限数量细胞的人类样本中的免疫状态提供了独特的机会。值得注意的是，我们测试了该面板与进一步下游应用的细胞分选兼容。此外，为了促进这种面板在不同群体和样本中的广泛实施，我们建立了一个精简的 45 色版本，可用于不同的光谱细胞术平台。最后，为了生成该面板，我们不仅利用了现有的面板设计指南，还开发了新的指标来系统地识别 50 种荧光染料的最佳组合，并在 50 色分离矩阵的背景下评估荧光染料特定的分辨率。
We report the development of an optimized 50‐color spectral flow cytometry panel designed for the in‐depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide‐spread implementation of such a panel across different cohorts and samples, we established a trimmed‐down 45‐color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome‐specific resolution in the context of a 50‐color unmixing matrix.