Multiparametric Flow Cytometry of Bone Marrow Cells

2 × 10⁶ MNCs were surface-stained for 15 min at 4°C or for 1 h at room temperature (RT) for intracellular staining with different combinations of antibodies (Table S1). DAPI (Molecular Probes, Eugene, USA) or LIVE/DEAD Fixable Blue Dead Cell Stain Kit (ThermoFisher Scientific) were used to exclude dead cells according to the manufacturer’s protocol. Cells were analyzed with a FACS Canto II or LSR Fortessa X-20 flow cytometer in standard configuration (BD Biosciences). PC, BC and T cells were identified as previously described (17 (link)) (Figure S1) and BMPC subsets were sorted. For quality control, Cytometer Setup & Tracking Beads (BD Biosciences) and SHPERO Calibration Particles (BD Biosciences) were used to get reproducible median fluorescence intensities (MFI). Staining controls were either done by fluorescence minus one (FMO) or isotype control approach.
For assessment of absolute counts, cells were resuspended in a defined volume after staining and 20 µl of CountBright^™ Absolute Counting Beads (ThermoFisher Scientific) were added before acquisition. Absolute cell counts were calculated as follows: