Mass Cytometry Staining and Data Acquisition
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PBMCs were isolated by Ficoll-Paque density-gradient centrifugation and cryopreserved in liquid nitrogen until time of analysis in RPMI, 20%FCS, 10%DMSO. Two metal conjugated 40-antibody panels for mass cytometry were developed, panel 1 focusing on B cell, NK cell and T cell markers and panel 2 focusing on myeloid and NK cell markers. Heavy metal isotope-tagged monoclonal antibodies (mAbs) for mass cytometry are listed in PBMCs were thawed, washed with RPMI, 50%FCS, and incubated with 0.04 mg/mL DNase in IMDM, 10% FCS in at room temperature (RT) for 30 min. Cells were washed with IMDM, 10% FCS, counted and for each panel 2.5*10Cells were acquired within 48 h of staining on a Helios mass cytometer (Fluidigm) at an event rate of <250 events/sec in Cell Acquisition Solution containing ×10 diluted EQ Four Element Calibration Beads (Fluidigm). For the compensation matrix, staining beads (eComp) were individually stained with the conjugated mAbs and incubated for 45 min. After washing, the beads were pooled, washed and acquired in cell staining buffer. Experiments and acquisition were performed in a period of 81 days.