Protocol for profiling circulating T cell subsets, their homing and regulatory markers, in pediatric samples using a 4-laser BD LSRII

Here, we present a protocol for profiling T cell subsets, regulatory phenotypes, and gut- and skin-homing markers of unstimulated cells or those stimulated with staphylococcal enterotoxin B (SEB), lipopolysaccharide (LPS), or zymosan (ZYM). We describe steps for isolation, cryopreservation, thawing, stimulation, and multiparameter flow cytometric analysis of human peripheral blood mononuclear cells (PBMCs). Use of the multicolored panel detailed in this protocol allows full utilization of the valuable samples with the minimal possible volume of collected pediatric blood. For complete details on the use and execution of this protocol, please refer to Thon et al. 1