Cd16 bv510 3g8

For each participant, 2.5–5 × 10⁶ PBMC were stained in 100uL FACS Buffer (PBS, 5% FCS, 0.016% EDTA) in 96-well plates. PBMC were first incubated with an amine-reactive viability dye (LIVE/DEAD Aqua, Invitrogen) in PBS, washed twice, and then stained with a combination of fluorescently-tagged antibodies in FACS buffer: CD19-BUV395 (SJ25C1), CD5-BV785 (UCHT2), and CD21-PE (B-ly4) from BD Biosciences and IgD-AF488 (IA6–2), IgM-PacBlue (MHM-88), CD27-PEDazzle (O323), CD38-AF700 (HIT2), CD24-PECy7 (ML5), CD10-PECy5 (HI10a), CD3-BV510 (OKT3), CD14-BV510 (M5E2), and CD16-BV510 (3G8) from BioLegend. Cells were then washed twice in FACS Buffer and refrigerated until analysis on the BD LSRII (initial cohort) or BD Fortessa (expansion cohort) at the Vanderbilt Flow Cytometry Shared Resource on the same date. The initial cohort used for tSNE analysis (5 healthy controls and 11 SSc patients) were collected as a single batch. The validation cohort containing an additional 11 healthy controls and 9 SSc patients were run at a later date and included only in biaxial gating analysis. For biaxial analysis forward and side scatter properties were used to identify single cells, then live B cells were identified by CD19 expression and exclusion of the viability dye. T cells, monocytes, and NK cells were removed from analysis by excluding CD3+, CD14+, and CD16+ events.