Comprehensive PBMC Phenotyping by Mass Cytometry

Cryopreserved PBMC were thawed by incubating cryovials at 37°C for 1 min and stained with the viability marker Cisplatin. Subsequently, PBMCs were incubated with an antibody mixture for 30 min. After washing, PBMCs were fixed in PBS (Thermo Fisher) with 2% PFA (Sigma-Aldrich) overnight at 4°C. The next day, PBMCs were stained with an intracellular antibody mixture after permeabilization using saponin-based Perm Buffer (eBioscience). After washing, cellular DNA was labeled with Cell-ID Intercalator-Ir (Fluidigm) and cell pellets were resuspended in 1:10 EQ Beads (Fluidigm) in 1 mL MiliQ water. Samples were measured using a Helios mass cytometer (Fluidigm). Twenty One different PBMC cell subsets were identified using the unsupervised gating approach DAFi⁶⁰ (link) with the exception of antibody-secreting cells (ASCs), which were manually gated as CD45⁺Live⁺CD14⁻CD3⁻CD19⁺CD20⁻CD38⁺ cells. Gating was performed using FlowJo (BD, version 10.7.0).