High Throughput Subpopulation Signaling Analysis: Protocol Considerations For Multiplexed Intracellular Flow Cytometry

Abstract While much of our knowledge of signaling pathways has come from bulk analytical methods such as western blotting, more granular techniques enable us to deepen our understanding of how signaling profiles may vary in heterogeneous cell populations. Flow cytometry offers a high-throughput avenue for subpopulation signaling analysis. Combining antibodies against extracellular CD markers with those against intracellular signaling proteins or post-translational modifications enables researchers to explore variations in signaling profiles by cell type. Performing a staining protocol that allows for accurate performance of antibodies against both extracellular and intracellular epitopes is key. Manufacturers commonly validate antibodies using specific protocols; straying from these may lead to drastically different experimental results and conclusions. Antibodies against CD markers, for example, are often validated for use on live cells, and may perform poorly on fixed and permeabilized cells. Compromised CD marker detection may lead to inaccurate gating and thus inappropriate subpopulation analyses. In turn, this can lead to anomalous conclusions about the signaling profiles of subpopulations of interest. In order to mitigate the risk of such occurrences, we present a general guide to protocol considerations when performing multiplexed intracellular flow cytometry through the lens of immune cell stimulation. Highlighting subpopulations of primary mouse immune cells treated with 2′3′-cGAMP, we demonstrate that CD marker staining protocol variation can lead to erroneous subpopulation characterization, which can then impact interpretation of innate immune signaling profiles within these populations.