CyTOF Barcoded Sample Batch Processing and Analysis

Barcoded sample batches were processed simultaneously to reduce data collection variability. Sample barcoding and data acquisition were carried out as previously described (18 (link)). Briefly, cells were washed twice in Maxpar® Barcode perm buffer (Fluidigm; US) and each sample was stained with a different barcode combination using Cell-ID 20-plex Pd-barcoding kit (Fluidigm; US) for 30 min at room temperature. After washing twice in CSB, samples were pooled together into one tube. Cells were incubated for 20 min at room temperature with 125 nM Cell-ID™ DNA Intercalator-Ir191/193 (Fluidigm; US), washed twice with CSB and once with Di water and lastly resuspended with 0.1 % EQ four element calibration beads (Fluidigm; US). Data were acquired with a CyTOF2® instrument (Fluidigm; US). After acquisition, data were concatenated, normalized using mass bead signal and de-barcoded using the CyTOF 2 software.