Comprehensive CyTOF Immunophenotyping Protocol

The flow CyTOF experiments were performed as described previously (57 (link)). Briefly, after dissociated cells were barcoded according to the manufacturer’s protocol (Fluidigm, 101-0804 B1), they were labeled with 36 metal-conjugated antibodies in FoxP3 permeabilization buffer (eBioscience, 00-8333) with 1% FBS (Hyclone, catalog 7207) for 12 hours at 4°C at a concentration of up to 3 million cells per 300 μL antibody cocktail, followed by washing twice with FoxP3 permeabilization buffer. Cells were then incubated with the DNA intercalator iridium (Fluidigm, 201192A) at a dilution of 1:4000 in 2% paraformaldehyde (Electron Microscopy Sciences, 15714) in Dulbecco’s PBS (Corning, 21-031-CV) at room temperature for 1 hour. Mass cytometric data were acquired on a Fluidigm Hyperion instrument. Flow CyTOF data analyses of endocrine and immune cell composition were performed using the Cytobank platform (https://www.cytobank.org/).