Phospho flow cytometry provides detailed insight into signaling events. (A) Phospho flow cytometry workflow overview. PBMCs were stimulated with PGE2 (0.01, 0.1, 1, and 10μM) over a 60-minute time period (0, 1, 3, 10, 30, and 60 minutes) in the absence and presence of the PKA inhibitor H89 (20μM). All samples were then fixed, subjected to fluorescent cell bar coding (FCB), and combined in 1 sample tube. Cells were then permeabilized and labeled with phospho-epitope–specific antibodies and cell type-specific surface marker antibodies before flow cytometric analysis and data analysis with the use of Cytobank (https://cytobank.stanford.edu). (B-C) Phospho flow cytometry results from 1 representative donor of 3 depicted as a heatmap. Warmer colors (yellow) indicate an increase in phospho-epitope–specific antibody signal, whereas colder colors (blue) indicate a decrease in signal. Differences in antibody signal were calculated with the first column of each lane as reference.
