Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studiesc - Mass cytometry, staining Panel A

This .fcs files form the part of the manuscript titled “Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studies”. The .fcs files presented in this experiment were obtained for mass cytometry where antibody panel A was used. Herein we have evaluated two whole blood preservation protocols that allow rapid sample processing and long-term stability for mass cytometry experiments. Two fixation buffers were used, Phosphoflow Fix and Lyse (BD) and Proteomic Stabilizer (PROT) to fix and freeze whole blood samples for up to 6 months. Both surface and intracellular, cytokine markers were evaluated.
Conclusion:
Whole blood can be successfully preserved for phenotyping and cytokine-response studies provided a careful selection of a compatible antibody panel. Our settings constitute a valuable tool for multicentric and retrospective studies.
Notes:
For MC experiments 500 ul blood aliquots were incubated for 6h at 37°C at 7% CO2 with the TLR7/8 ligand resiquimod, 1.25ug/ml (R848, Invivogen) in the presence of Protein Transport Inhibitor Cocktail 1X (Thermo Fisher Scientific). The samples were stained immediately (FRESH), or fixed using BD or PROT protocols. Raw data were normalized using CyTOF software 6.7 (Fluidigm). FlowJo software v10.0 was used to analyze normalized MC data by manual gating. Live leukocytes were selected using Pt staining and extracted to FCS files. Two selected functions from the flowAI package (flow_rate_bin, flow_rate_check) were used to remove regions with unstable flowrate. To make this function applicable to MC data, we adapted the TIMESTEP to 1 bin per 10 seconds. flowCut algorithm was used to remove signal instability with 1000 segments and MaxPercCut to 0.5. These files contain normalized, cleaned and live leukocytes events. Detailed gating strategy can be found in figure 3 in the manuscript.