OMIP 082: A 25‐color phenotyping to define human innate lymphoid cells, natural killer cells, mucosal‐associated invariant T cells, and γδ T cells from freshly isolated human intestinal tissue

Abstract

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.

OMIP‐096: A 24‐color flow cytometry panel to identify and characterize CD4+ and CD8+ tissue‐resident T cells in human skin, intestinal, and type II mucosal tissue

“…This non‐specific cleavage of markers requires a series of optimization steps prior to panel development to be confident that the cells that are acquired are similar to those (in situ) within the tissue. A significant novelty of this publication is the application of our optimization protocols that minimize the effects of enzymatic cleavage on surface proteins [ 6 , 9 , 10 ]. This is primarily performed through the identification of the affected markers, and testing multiple antibody clones and alternative enzymes (Figures S3 and S4 ).…”