Differentiation and detection of mouse CD4+ Th cells by measuring intracellular cytokine production (62.6)

Abstract

Abstract Naïve CD4+ T cells can give rise to a variety of subsets of Th cells depending on the nature of the immune response, and subsequently release a distinct subset of cytokines. Conventionally these cytokines can be measured in a secreted format (e.g. Luminex or ELISAs), but here we describe a novel method to reliably differentiate mouse CD4+ T cells, and further characterize the differentiated lineages by measuring intracellular cytokines using flow cytometry. In addition, we also describe the use of a proprietary fixable viability dye in order to eliminate the false positives associated with non-specific staining. Based on an optimized protocol for differentiating CD4+ T cells, we have developed differentiation tools designed to obtain the desired Th cell lineages. Following naïve T cell differentiation toward specific Th lineages, we further developed a flow cytometry assay to measure intracellular cytokine production by blocking the secretion of the cytokine with Brefeldin A. We further eliminate non-specific staining due to dead or dying cells using a proprietary fixable viability dye and accurately determine the percentage of Th lineages using guava benchtop flow cytometer and InCyte analysis software.