Anti cd38 pe

Cells were treated with 5 mM EDTA in PBS before staining, which reduced the binding of cytophilic nonendogenous Abs to B cells (Supplemental information). Cells were treated with unlabeled anti-CD16/CD32 (2.4G2, eBioscience) in FACS buffer (1% BSA/PBS) to block FcγRs. After washing, cells were stained with the relevant antibodies: anti-Igκ-FITC (187.1, BD Pharmingen), anti-IgM-PerCP-Cy5.5 (II/41, eBioscience), anti-IgD-FITC (11–26 c.2a, BD Pharmingen), anti-IgG1-PE (A85-1, eBioscience), anti-CD45R/B220-PE-Cy7 (RA3-6B2, eBioscience), anti-CD138-Biotin (281.2, BD Pharmingen), anti-GL7-FITC (GL7, BD Pharmingen), anti-CD38-PE (90, eBioscience), anti-CD95/Fas-PE (Jo2, BD Pharmingen), anti-CXCR4-APC (2B11, eBioscience), and PE–streptavidin (eBioscience). Cells were analyzed on FACSCanto II and FACSAria II (BD Biosciences). Cell sorting was performed on FACSAria II (BD Biosciences). The data were analyzed using FlowJo software (Tree Star).

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).