Cd5 percp

Peritoneal cells were subjected to flow cytometry analysis using the antibodies CD19-Alexa Fluor 647 (rat anti-mouse IgG2a, clone: 6D5; BioLegend, San Diego, California, USA), CD5-PerCP (rat anti-mouse IgG2a, clone: 53–7.3; BD Pharmingen, San Jose, California, USA), and CD11b-PE (rat anti-mouse IgG2b, clone: M1/70; BD Pharmingen, San Jose, California, USA). Measurement was made using a CyAn ADP flow cytometer (Dako, Glostrup, Denmark). B-1a cells were characterized as CD19⁺CD5⁺CD11b⁺, B-1b cells as CD19⁺CD5^-CD11b⁺, and B-2 cells as CD19⁺CD5^-CD11b^-, using the CyAn ADP flow cytometer and Summit software version 4.3 (Dako, Glostrup, Denmark). Rat IgG2a FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) and rat IgG2b FITC-conjugated isotype control (Pierce, Thermo Scientific, USA) were used as the isotype controls.

The following antibodies were used for staining: anti-mouse IgM-APC, B220-FITC, CD93-PE CF594, CD19-Alexa Fluor 647, IL10-PE, CD23-BB515, CD5-PerCP, CD1d-BV421, CD43-PE Cy7, B220-APC Cy7, Fc Blocker (CD16/CD32), Cytofix/CytoPerm kit (all from BD Pharmingen), CD23-PerCP/Cy5.5, CD93-PE Cy7, CD1d-PB, CD5-PE Cy7, CD21-APC Cy7, CD19-PE, CD43-APC Cy7 (all from Bio Legend), Live/Dead aqua marker (Molecular Probes).