Determining intracellular cytokine expression and viability simultaneously in differentiated mouse CD4+ T-cells through flow cytometry (152.8)

Abstract

Abstract The study of CD4+ T-helper (TH) cell differentiation is an important area of research that will aid in the understanding of inflammation and autoimmunity. CD4 + T cells can give rise to many subtypes depending on type of immune response. This study focuses on the TH1, TH2 and TH17 CD4+ T cell subtypes. Each subtype expresses a signature cytokine that directs the type of immune response needed. In order to analyze cytokine expression in TH cultures by flow cytometry, we employed a fixable viability dye to gate out dead cells that can accumulate in long-term differentiation cultures. In addition, flow cytometric analysis provides cell-specific information not obtained by ELISA analysis. Using standard TH culture protocols we differentiated naïve CD4+ T cells into TH1, TH2, and TH17 subtypes. We first stained the cells with the viability dye and then stained with antibodies against the signature cytokine of interest in less than 4 hours. Our data shows that we can easily obtain viability information while simultaneously evaluating cytokine production within our TH culture system. Using the fixable viability dye, we can exclude false positive cytokine staining and therefore obtain more accurate and reproducible intracellular expression data from cultured CD4+ T cells.