An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells

To evaluate the robustness of a 3 panel FC barcoded phosphoflow panel for the measurement of ERK1/2, NFkB p65, p38, Stat1 S727, Stat1 Y701, Stat3 S727, Stat3 Y705 in monocytes, T cells, NK cells, CD3+CD56+ cells and B cell. Analysis was conducted using cryopreserved PBMC from 3 donors, with 3 repeat measures conducted over a 1 month period.
Conclusion:
The data indicated excellent precision of measurements (as assessed by the coefficient of variation, calculated against the 3 replicant). The robustness of the assay was shown to vary based on stimulus, cell type and phospho-protein measured; but in most instances were found to be lower than 10%.
Notes:
9x barcoding stimulus combinations are as follows- POlow + PB low= Unstimulated, POlow + PBmid= IFNalpha (100ng/ml), POlow + PBhigh= IL-2 (100ng/ml), POmid + PBlow= IL-6 (100ng/ml), POmid + PBmid= PMA (100ng/ml), POmid + PBhigh = IL-10 (100ng/ml), POhigh + PBlow = IFNgamma (100ng/ml), POhigh + PBmid = LPS (10micrograms/ml), and POhigh + PBhigh= Unstimulated. Samples acquired on a LSRI Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). CS & T beads were used daily to assess consistency of machine measurements.