The Challenge of Distinguishing Cell-Cell Complexes from Singlet Cells in Non-Imaging Flow Cytometry and Single-Cell Sorting.
The Challenge of Distinguishing Cell-Cell Complexes from Singlet Cells in Non-Imaging Flow Cytometry and Single-Cell Sorting.
复制标题
DOI：
10.1002/cyto.a.24027
复制
发表时间：
2020-11
期刊：
影响因子：
--
通讯作者：
Peters B
中科院分区：
文献类型：
--
作者：
Burel JG;Pomaznoy M;Lindestam Arlehamn CS;Seumois G;Vijayanand P;Sette A;Peters B
我们最近的工作强调了在解释来自流式细胞术采集或细胞分选的单细胞数据时需要小心：我们发现与其他免疫细胞结合的T细胞的双峰通常存在于通过流式细胞术采集的人外周血样本的活单态门中。这种隐藏的“污染”在被认为是单细胞的细胞中产生混合细胞谱系的非典型基因特征，这可能导致数据误解，例如描述新的免疫细胞类型。在这里，基于T细胞-单核细胞复合物的例子，我们确定了实验和数据分析策略，以帮助在非成像流式细胞术和单细胞分选中区分单细胞和细胞-细胞复合物。我们在T细胞-单核细胞和T细胞-B细胞复合物中发现了强大的分子特征，可以在蛋白质和mRNA水平上将它们与单线态区分开来。采用适当的设门策略（与细胞分选中使用的设门策略相匹配）的成像流式细胞术和细胞分选后的直接显微镜成像是检测可疑双重表达细胞中细胞-细胞复合物存在的两种首选方法。最后，我们应用这些知识来强调T细胞-B细胞复合物在最近发表的数据集中的可能存在，该数据集描述了具有混合T细胞和B细胞谱系特性的新型细胞群体。版权所有2020作者。Cytometry Part A由Wiley Periodicals，Inc.出版。代表国际细胞计数促进协会。
Our recent work has highlighted that care needs to be taken when interpreting single cell data originating from flow cytometry acquisition or cell sorting: We found that doublets of T cells bound to other immune cells are often present in the live singlet gate of human peripheral blood samples acquired by flow cytometry. This hidden “contamination” generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types. Here, based on the example of T cell–monocyte complexes, we identify experimental and data analysis strategies to help distinguishing between singlets and cell–cell complexes in non‐imaging flow cytometry and single‐cell sorting. We found robust molecular signatures in both T cell–monocyte and T cell–B cell complexes that can distinguish them from singlets at both protein and mRNA levels. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two methods of choice to detect the presence of cell–cell complexes in suspicious dual‐expressing cells. We finally applied this knowledge to highlight the likely presence of T cell–B cell complexes in a recently published dataset describing a novel cell population with mixed T cell and B cell lineage properties. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
登录
查看更多内容
影响因子：
30.5
作者：
通讯作者：
--
影响因子：
4.4
作者：
Daubeuf, Sandrine;Lindorfer, Margaret A.;Hudrisier, Denis
通讯作者：
Hudrisier, Denis
影响因子：
48
作者：
Picelli, Simone;Bjorklund, Asa K.;Sandberg, Rickard
通讯作者：
Sandberg, Rickard
影响因子：
24.8
作者：
Patil VS;Madrigal A;Schmiedel BJ;Clarke J;O'Rourke P;de Silva AD;Harris E;Peters B;Seumois G;Weiskopf D;Sette A;Vijayanand P
通讯作者：
Vijayanand P
DOI：
10.1126/science.aan6828
发表时间：
2017-10-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Stubbington MJT;Rozenblatt-Rosen O;Regev A;Teichmann SA
通讯作者：
Teichmann SA