Using Full‐Spectrum Flow Cytometry to Phenotype Memory T and NKT Cell Subsets with Optimized Tissue‐Specific Preparation Protocols

Abstract Full‐spectrum flow cytometry is now routinely used in many laboratories internationally, and the demand for this technology is rapidly increasing. With capacity to use larger and more complex staining panels, standardized protocols are required for optimal panel design and analysis. Importantly, for ex vivo analysis, tissue preparation methods also need to be optimized to ensure samples are truly representative of tissues in situ . This is particularly relevant given the recent interest in adaptive immune cells that form residency in specific organs. Here we provide optimized protocols for tissue processing and phenotyping of memory T cells and natural killer T (NKT) cell subsets from liver, lung, spleen, and lymph node using full‐spectrum flow cytometry. We provide a 21‐color antibody panel for identification of different memory subsets, including tissue‐resident memory T (T RM ) cells, which are increasingly regarded as important effectors in adaptive immunity. We show that processing procedures can affect outcomes, with liver T RM cells particularly sensitive to heat, such that accurate evaluation requires fast processing at defined temperatures. © 2022 Wiley Periodicals LLC. Basic Protocol 1 : Processing mouse liver for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 2 : Processing mouse spleen for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 3 : Processing mouse lungs for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 4 : Processing mouse lymph nodes for flow cytometric analysis of memory T and NKT cell subsets Basic Protocol 5 : Staining and flow cytometric analysis of samples for memory T and NKT cell subsets Support Protocol : Obtaining cell counts from flow cytometry data