Multiparametric Profiling of Immune Cells

Spleen and lung cells from mice, and human PBMCs were first stained with 200 µM cisplatin (Sigma) for the discrimination of dead cells. Upon this, cells were washed and stained with a primary antibodies cocktail for 30 minutes at 4 °C (Supplementary Table 1 (for human antibodies) and Supplementary Table 5 (for mouse antibodies)). Dilution of different antibodies used was either 1:100 or 1:200 and was standardized by our CyTOF platform. After washing, cells were stained with the heavy metal-labeled antibodies cocktail for 30 minutes at 4 °C. Cells were washed, fixed and permeabilised using fixation FoxP3 buffer (eBioscience) for 45 minutes at 4 °C. Upon this cells were stained with intranuclear antibodies cocktail for 30 minutes at 4 °C. Labeled cells were washed twice with PBS and fixed in 2% or 4% PFA overnight. Subsequently, cells were washed and stained for barcodes (30 minutes at 4 °C) and DNA (10 minutes at 4 °C). Samples were acquired on the Helios CyTOF (Fluidigm), and analyzed as mentioned before^{24 ,25 (link),71 (link)}. For details, see supplementary methods.