Helios mass cytometry

Fresh blood was processed and stained using the Maxpar Direct Immunoprofiling Assay (MDIPA) (Fluidigm or The Longwood Medical Area CyTOF core, Boston, MA). Following processing and staining, samples were stored at −80° C. The frozen stained blood samples were placed at room temperature (RT) for 30–50 min until fully thawed and then incubated with 1× Thaw-Lyse buffer (Smart Tube Inc.) at RT for 10 min. The lysis steps were repeated up to 3 times until the pellet turned white evidencing complete lysis of red blood cells. The cells were then incubated with Maxpar Fix and Perm Buffer (Fluidigm) containing 125 nM Cell-ID Intercalator-Ir solution (Fluidigm) at 4° C overnight or up to 48 h before sample acquisition. Groups of samples (4–8/day) were assessed by Helios mass cytometry (Fluidigm) in 4 independent experiments using a flow rate of 45 μI/min in the presence of EQ Calibration beads (Fluidigm) for normalization. An average of 400,000 ± 50,000 cells (mean ± SEM) from each sample were acquired and analyzed by Helios. Gating was performed on the Cytobank platform (Cytobank, Inc. Santa Clara, CA) and FlowJo 10.5.3 (BD Biosciences).

Start the mass cytometry (Helios Mass Cytometry, FLUIDIGM), debug instruments and control quality standards (Tuning Solution, FLUIDIGM), the same number of cells in each sample were mixed together, add calibration beads (EQ™ Four Element Calibration Beads, FLUIDIGM), set channel name, data name, collection speed, and total cell number, and collect the raw data.