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 frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL‐2 and 21% for and INF‐γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL‐2 and 32% for INF‐γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL‐2 and IFN‐γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL‐2 and IFN‐γ mRNA and protein expression in frozen blood

The Fluorescence, Flow Cytometry and Cytomics symposium welcomes you to this excursion into instrumental methods of deciphering these inner galaxies of biology (with due apology to the late Douglas Adams) [1]. It has usually been our intention to review some of the instrumental improvements in flow cytometry, multiparameter fluorescence detection and correlative aspects of microscopy with confocal and other laser or fluorescence microscopies [2]. We will continue to do this, and we will have excellent instrumental presentations. It is time to improve our perspective of just what we are looking for. There is grandeur in these views of life, even from the microscopical view alone, and these universes only get better as instruments improve.