Anti cd24 alexa fluor647

Flow cytometry was performed with the following antibodies: anti-CD19-Percp-Cy5.5, anti-CD19-Percp-eFluor710, anti-CD24-FITC, anti-CD38-superbright600, anti-CD38-PE, anti-Ki-67-Alexa Fluor647, anti-IL-10-PE, anti-TIM-1-PE, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-17A-APC (eBioscience, San Diego CA, USA), anti-IL-6-APC, anti-TNF-α-FITC, anti-IL-12-BV-421, and anti-CD24-Alexa Fluor647 (BD Biosciences, France). Intracellular cytokines and Ki-67 were assessed in cells treated with Permeabilization and IC Fixation Buffers (eBioscience, San Diego, CA, USA). Stained cells were analyzed with a 21-color ZE5 cell analyzer (Bio-Rad, USA). The CD19⁺CD24^hiCD38^hi B subset was sorted (FACSAria; BD Pharmingen) using anti-CD19-Percp-Cy5.5, anti-CD24-FITC, and anti-CD38-PE. The sort purity of CD19⁺CD24^hiCD38^hi B cells was routinely >90%. CD4⁺T subpopulation was sorted (FACSAria; BD Pharmingen) using anti-CD38-PE and anti-CD3-PE. The sort purity of CD4⁺T cells was routinely >95%. All flow cytometry data were analyzed with FlowjoV10 Software (Tree Star, OR, USA).

B cells enriched cell fractions were stained with the following combination of mAbs: anti-IgD Alexa Vio770 (BioLegend, San Diego, CA, USA); anti-IgM PerCP_Cy5.5, anti-CD27 PE-CF594, anti-CD38 PE-Cy7, anti-CD24 Alexa Fluor 647 and anti-CD5 BV 421, anti-IGκ FITC, anti-IGλ PE, anti-IgA VioGreen (BD). B cell subsets were isolated by FACS sorting (FACSAria, Becton Dickinson, Franklin Lakes, NJ, USA) after depleting IgA⁺ and dead cells with a two-step sorting approach: 1) a four-way pre-sort with yield setting was used to separate enriched B cells into IGκ⁺/CD5⁺, IGκ⁺/CD5^-, IGλ⁺/CD5^+,and IGλ⁺/CD5^-B cells; 2) each of the above cell fractions were then sorted into six main B cell subpopulations (after excluding CD38^highCD24^- plasmablasts): CD24^highCD38^high transitional (TR), IgD^highIgM⁺CD38^-CD27^- naive (N), IgD^low IgM⁺CD38^-CD27⁺ marginal zone-like (MZ), IgM⁺IgD^-CD38^-CD27⁺ IgM-only memory (MO), IgM^-IgD^-CD38^-CD27⁺ switch-memory (SM), and IgM^-IgD^-CD38^-CD27^- double negative (DN) B cells. See also Figure 1 and Supplementary Figure S1 for details.