Effective detection of phosphorylated Stat5 in FoxP3-positive regulatory T cells (121.24)

Abstract

Abstract Multicolor flow cytometric analysis is an invaluable tool for analyzing unique cell types within heterogeneous primary cell samples, like peripheral blood mononuclear cells (PBMCs). To identify these specific populations, antibodies against cell surface markers are typically used. However, obtaining greater in-depth knowledge often requires analysis of intracellular events, like phosphorylation of signaling molecules or expression of transcription factors. A challenge to staining cells for both cell surface and intracellular markers is the lack of compatibility of the cell fixation and permeabilization agents with the antibodies required for cell surface and intracellular staining. In this report, we developed an optimized protocol for the simultaneous detection of the cell signaling molecule Stat5 together with transcription factors and cell surface markers. We evaluated human PBMCs for phospho-Stat5 signal induction in Treg cells upon stimulation with IL-2. Using the optimized buffer protocol with antibodies at their optimal concentrations for this intracellular staining condition, we detected the phosphorylation of Stat5 in CD4+/CD25bright/FoxP3+ Treg cells in response to IL-2 stimulation. We also observed differential phosphorylation of Stat5 in various lymphocyte subsets, including memory and naive T cells, when human whole blood was stimulated with IL-2, lysed, fixed, permeabilized, and stained for CD3, CD4, CD45RA, CD45RO, T-bet, and pStat5 expression.