Cd24 fitc ml5

Abs used were CD19 PE (LT19; Miltenyi Biotec), CD138 allophycocyanin (B-B4; Miltenyi Biotec), CD38 PE-Cy7 (HB7; BD Biosciences), CD38 AF700 (HIT2; BioLegend), CD20 eFluor V450 (2H7; eBioscience); CD27 AF647 (LT27; AbD Serotec), CD27 FITC (M-T271), CD19 PerCP-Cy5.5 (SJ225C1; BD Biosciences), CD19 PE-Cy7 (SJ225C1; BD Biosciences), CD24 FITC (ML5; BD Biosciences), CD84 PE (CD84.1.21; BioLegend), CD38 PerCP-Cy5.5 (HIT2; BD Biosciences), CD95 BV421 (DX2; BioLegend), CD20 allophycocyanin-H7 (L27; BD Biosciences), CD27 BV605 (O323; BioLegend), CD3 VioGreen (BW264/56; Miltenyi Biotec), Ki67 FITC (B56; BD Biosciences), unconjugated goat anti-IRF4 (M-17; Santa Cruz Biotechnology), and donkey anti-goat IgG AF488 (polyclonal; Invitrogen). Controls were isotype-matched mouse mAbs. Annexin V FITC was from eBioscience, and 7-AAD was from BD Biosciences.
Reagents included human IL-2 (Roche), IL-6 (PeproTech), IFN-α (Sigma), IL-21 (PeproTech), goat anti-human F(ab′)₂ fragments (anti-IgM and anti-IgG; Jackson ImmunoResearch), HybridoMax hybridoma growth supplement (Gentaur), Lipid Mixture 1, Chemically Defined (200×), and MEM Amino Acids Solution (50×, Sigma).

Cells were analysed using seven‐colour direct immunofluorescence staining on a CytoFLEX LX Flow Cytometer (Beckman Coulter). Abs used were CD20‐AF700 (clone 2H7), CD27‐PE (M‐T271), CD38 PE‐Cy7 (HB7), CD24‐FITC (ML5) and IgD‐V500 (IA6‐2), all from BD Biosciences with CD138‐VioBlue (44F9) (Miltenyi). In a second panel, CD20, CD27, CD38, CD138 and IgG‐V500 (G18‐145); IgM‐APC (G20‐127) (both BD Biosciences); and IgA‐FITC (IS11‐8E10) (Miltenyi) were also tested on circulating B cells (day 0 only). Cell populations were gated on FSC and SSC plot to remove small debris events (including apoptotic bodies), while a total event gate recorded viable and dead cells (gated using the 7AAD dye 50 µg/ml [BD Biosciences]). Absolute cell counts were calculated using CountBright beads (25,000 beads/panel; Invitrogen). Analysis was performed with CytExpert software, version 2.3 (Beckman Coulter).
A classic gating strategy for positive/negative live cell events was used (see profile in Figure S1) to evaluate changes in marker positivity at days 0, 6 and 13 and reported as % of total live cells. A gating strategy to define circulating naïve/memory from B‐reg and EPB, and expression of surface Ig is described in Figure S2.