Department of Pediatrics, Division of Infectious and Immunological Diseases, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.
Cytometry A. 2010 Jun;77(6):546-51. doi: 10.1002/cyto.a.20862.
Polychromatic flow cytometric analysis takes advantage of the increasing number of available fluorophores to positively identify and simultaneously assess multiple parameters in the same cell (1). Additional parameters may be analyzed through negative identification (i.e., through exclusion of particular stains or antibodies employed). In this report, we tested whether such negative-gating strategy would identify human B lymphocytes in innate immune phenotyping studies. To this end, B cells were identified as the negatively-stained subpopulation from the CD123 vs. CD11c plot of the CD14(neg-low), MHC II(high) human peripheral blood mononuclear cells. To test the specificity of this negative gating approach, we confirmed that negatively gated B cells indeed expressed CD19, the bona fide marker for human B cells. However, a small number of unidentified cells were contained in the negatively-gated B cells. Furthermore, a small percentage cells expressing markers used to identify monocytes and myeloid dendritic cells (mDC) coexpressed CD19. This identifies such negative B-cell gating approach as potentially problematic. When applied to the analysis of Toll-like receptors (TLR) stimulation experiments, we were however able to interpret the results, as B-cells respond to TLR stimulation in a qualitative different pattern as compared to monocytes and DC. This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) (2) and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt-compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data.
多色流式细胞术分析利用越来越多的荧光染料，对同一细胞进行多种参数的正向鉴定和同时评估（1）。通过负向鉴定（即通过排除特定的染色剂或抗体），还可以分析额外的参数。在本报告中，我们测试了这种负向门控策略是否可以在固有免疫表型研究中识别人类 B 淋巴细胞。为此，通过 CD14(neg-low)、MHC II(high)人外周血单核细胞的 CD123 与 CD11c 图，将 B 细胞鉴定为负染亚群。为了测试这种负向门控方法的特异性，我们确认了负向门控 B 细胞确实表达了 CD19，这是人类 B 细胞的真正标志物。然而，在负向门控的 B 细胞中包含了少量未识别的细胞。此外，一小部分表达用于识别单核细胞和髓样树突状细胞 (mDC)的标志物的细胞也表达 CD19。这表明这种负向 B 细胞门控方法可能存在问题。然而，当应用于 Toll 样受体 (TLR)刺激实验的分析时，我们能够解释结果，因为与单核细胞和 DC 相比，B 细胞对 TLR 刺激的反应模式是定性不同的。本报告完全符合流式细胞术实验最低信息（MIFlowCyt）标准（2），最近被国际流式细胞术协会（ISAC）采用，并纳入了 Cytometry 和其他期刊的出版政策。我们展示了如何用最小的努力编写符合 MIFlowCyt 标准的报告，同时为读者提供更清晰的流式细胞术实验和数据图像。
