Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry.

Single-Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry. Curr Protoc Cytom. 2020 Mar;92(1):e69 Authors: Pal R, Schaubhut J, Clark D, Brown L, Stewart JJ Abstract Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell-surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell-surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL-2, INF-γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL-2 and INF-γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and froz...
Source: Current Protocols in Cytometry - Category: Molecular Biology Tags: Curr Protoc Cytom Source Type: research