Comprehensive Immune Profiling of Atherosclerosis

Cells isolated from plaque specimens and paired PBMCs from the same patient were barcoded using a CD45-based approach and optimized for scarce clinical samples^{63 (link),64} , pooled, and stained with antibody panels. All antibodies were validated at the Human Immune Monitoring Center of the Icahn School of Medicine at Mount Sinai ^{65 (link)} and data deposited (https://flowrepository.org/id/FR-FCM-Z23S). See Supplementary Table 3 for a complete list of antibodies used in this study. Antibodies were either purchased pre-conjugated from Fluidigm, or purchased purified and conjugated in-house using MaxPar X8 Polymer Kits (Fluidigm) according to the manufacturer’s instructions. The samples were then washed and stained with cisplatin-195Pt or Intercalator Rh103 (Fluidigm, 201064 and 201103A) as a viability dye ⁶⁶ , washed, fixed, and permeabilized with BD Perm/Wash and Cytofix/Cytoperm buffers (BD Biosciences, 51–2090KZ and 51–2091KZ), and stored in freshly diluted 2% formaldehyde (Electron Microscopy Sciences) in PBS containing 0.125 nM Iridium 191/193 intercalator (Fluidigm, 201192) until acquisition. A limitation of our T cell focused panel consisted in the lack of anti-foxp-3 antibody, which limited the ability of dissecting the Tregs compartment in human atherosclerosis.