Assessing IL-2-Induced STAT5 Phosphorylation in Fixed, Permeabilized Foxp3(+) Treg Cells by Multiparameter Flow Cytometry

Assessing IL-2-induced phosopho-STAT5 (pSTAT5) content can reveal the cytokine responsiveness of individual T cells. Identifying distinct T cell subsets by nuclear transcription factors, such as Foxp3, and concurrently quantifying intracellular pSTAT5, however, has been technically challenging. Conventional Foxp3 staining buffers quench pSTAT5 signals, while commonly used pSTAT5 staining protocols fail to detect Foxp3. The current protocol resolves these issues by describing a procedure to assess IL-2-induced pSTAT5 contents in Foxp3(+) CD4 Treg cells using multiparameter flow cytometry. For complete details on the use and execution of this protocol, please refer to Waickman et al. (2020).