Cd38 bb515 hit2

Samples used for mass cytometry validation were 200 μL of whole blood that were fixed in 1-step Fix/Lyse Solution (eBioscience), and stored at −80 °C. Fixed cells were thawed, permeabilized using Permeabilization Buffer (eBioscience) and then stained with the following antibodies specific for epitopes insensitive to fixation, purchased from Biolegend unless otherwise indicated: CD38 BB515 (HIT2, BD Biosciences), CD4 BV510™ (SK3), CD8 PerCP/Cy5.5 (HIT8a), CD161 APC (HP-3G10, eBioscience), HLA-DR APC/Cy7 (L243), TCR Vα7.2 BV605™ (3C10), TCR γ/δ BV421™ (B1), Ki67 PE (Ki-67), and CD3 BV650™ (UCHT1).

Synovial cell suspensions and matched fresh PBMC samples were stained with Zombie UV® (BioLegend, San Diego, CA) for 15 min in serum-free PBS prior to washing in 2 mL PBS (500×g, 5 min). Cell surface was then stained for 30 min with an antibody cocktail consisting of; anti-CD45RO-BUV395 (UCHL1), PD1-BV421 (EH12.1), CD3-PerCP-Cy5.5 (SK7), ICOS-PE (DX29), CD8-Alexa Fluor® 647 (RPA-T8), CD4-Alexa Fluor® 700 (SK3), CD20-APC-H7 (L27), TIGIT-PE-Cy7 (A15153G), CD38-BB515 (HIT2) (all from BD Biosciences, Franklin Lakes, NJ), and CXCR5-PE/Dazzle™ 594 (J252D4; BioLegend, San Diego, CA) in Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ). Following washing in PBS + 2% FCS, cells were resuspended in PBS + 2% FCS ready for FACS acquisition and sorting on a BD FACSAria™ Fusion flow cytometer. Populations of interest were collected in 1.5 mL Eppendorf tubes prior to storage.