Sa apc cy7

Mononuclear cell surface markers were stained with the following fluorochromes: human hematopoietic lineage markers (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) APC (eBioscience), CD34 PE, CD38 Alexa Fluor 700, CD7 PE-Cy5, CD10 PE-Cy7, CD49f PerCP-Cy5.5, CD45RA Brilliant Violet 570, CD135-Biotin, CD127 (IL7Rα) PE-Cy7, SA-APC-Cy7 (BioLegend). Following surface marker staining, cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Tween-20 and stained with goat anti-human ARID3a antibody (18 (link)), followed by rabbit anti-goat FITC (Invitrogen). Isotype controls (BD Biosciences, eBiosciences, BioLegend) were used for gating hematopoietic progenitor subsets as described (22 (link), 24 (link)) . Doublet exclusion was used to ensure analyses of single cells prior to forward/side scatter gating. Data were collected using an LSRII (BD Biogenics) and FACSDiva (BD Biosciences) software version 4.1, and were analyzed using FlowJo (Tree Star) software version 10.

Spleens and adipose tissue (pooled gonadal/renal fat pads) were harvested and processed as described (5 (link), 6 (link)). Cells were stained with antibodies, then results were acquired using a BD LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star). CD19-BV510 and Vβ8.3-PE (BD Biosciences). I-A^d-A488, CD8α-A700, CD4-APC, CD11c-APC/Cy7, CD3ε-PE, CD11b-PE/Cy7, CD54-FITC, CD40-PE, H²K^d-FITC, I-A^d-PE, CD11c-biotin, SA-APC/Cy7 and TruStain FcX (BioLegend). CD86-APC (eBioscience).

For flow cytometry, cells were stained with mAbs in PBS with 1% BSA for 20 minutes in the dark at 4°C. Cells were subsequently washed and fixed with a 1% paraformaldehyde/PBS solution before analysis on BD FACSCalibur flow cytometer (BD Immunocytometry Systems, [BDIS] San Jose, CA) or MACSQuant® Analyzer (Miltenyi Biotec). The anti-human antibodies used were CD19 Pacific Blue, CD24 PE-Cy7, CD38 APC, CD27 APC-Cy7, CD21 FITC, CD1c PE (all from Biolegend, San Diego, CA), CD10 PE, CD5 PE, CD23 PE, IgD PE, IgM PE-Cy5 (all from BD Bioscience), BAFF-R PE (eBioscience Inc., San Diego, CA). The following anti-mouse antibodies were used: CD24 Pacific Blue, CD21 FITC, CD23 PE, BAFF-R PE, CD21 APC, SA-APC-Cy7 (secondary antibody for IgD biotin staining), IgM PE-Cy7 (all from Biolegend), and IgD Biotin (eBioscience). Living cells were identified by forward scatter and side scatter gating and/or exclusion of 7-aminoactinomycin-D (eBioscience) added immediately prior to data acquisition or fixation. Flow cytometry data analysis was performed using Flowjo data analysis software (TreeStar, Ashland, OR).