Doyle Chloe M, Fewings Nicole L, Ctercteko Grahame, Byrne Scott N, Harman Andrew N, Bertram Kirstie M
Centre for Immunology and Allergy Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.
Centre for Virus Research, The Westmead Institute for Medical Research, Westmead, New South Wales, Australia.
Cytometry A. 2022 Mar;101(3):196-202. doi: 10.1002/cyto.a.24529. Epub 2022 Jan 12.
We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.
我们开发了一个25色流式细胞术检测板，用于全面检测人体组织中的固有淋巴细胞（ILC）、黏膜相关恒定T（MAIT）细胞、自然杀伤（NK）细胞和γδT细胞。从新鲜人体组织中分离和检测这些细胞的能力，对于理解这些细胞在健康和疾病状态下在肠道等免疫特权黏膜表面所起的作用至关重要。然而，由于许多关键的表面识别标志物易受酶解作用影响，将这些细胞从组织中分离出来极具挑战性。因此，在高参数检测板中选择正确的酶 - 抗体克隆组合是一个关键考量因素。在此，我们对不同常见消化酶混合物对用于识别这些细胞类型的关键表面标志物的影响进行了全面、深入的分析。此外，我们比较了多种针对极易受酶解作用影响的表面标志物（如CD127和NKp44）的抗体克隆，以在供体之间实现最一致且优质的染色模式。
