Cd45r b220 biotin

For the isolation of T cells, splenocytes were labeled with CD8α-PE and anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The labeled cells were then purified through LS column (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4⁺ T cells were isolated by using the mouse CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD11c⁺ DCs were purified by using CD11c⁺ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). B220⁺ cells were purified by using CD45R/B220 biotin (BD biosciences, California, USA) and anti-Biotin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) from splenocytes of naïve C57BL/6 mice. Purified B cells were transduced with vacESAT6 at a MOI of 1 and/or loaded with 1 μg/ml of αGC and then co-cultured with naïve splenocytes for 24 h. Incubated cells were stained to examine the expression of B220, CD40, and CD86 using antibodies such as APC-conjugated anti-B220 (BD biosciences, California, USA), PE-conjugated isotype control (eBioscience, San Diego, USA), anti-CD40 (Biolegend, San Diego, USA), and anti-CD86 Ab (BD biosciences, California, USA). Cells were analyzed by flow cytometry.

Ten- to 12-week-old CXCR4-EGFP BAC transgenic reporter mice with or without LAD ligation were either treated with saline or DMOG (80 mg/kg/day) for up to 7 days. BM mononuclear and myocyte-depleted cardiac cells were separated as previously described [4 (link)]. Cells were incubated for 40 min in the dark at 4 °C with the following fluoresceinisothiocyanate (FITC)-, phycoerythrin (PE)-, and peridininchlorophyll-protein (PerCP)-conjugated monoclonal antibodies: CD45-PerCP, CD11b-PErCP, CD11b-PE, CD4-PE, CD20-PE, CD31-PE, CD34-PE, Flk-PE, CD86-PE, CD206-PE, F4/80-PE, CD133-PE, c-kit-PE, Sca-1-PE, CD3-biotin, CD45R/B220-biotin, CD11b-biotin, TER-119-biotin, and Ly-6G-biotin (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen) served as controls. Cells were analyzed by three-color flow cytometer using a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter). Each analysis included 50,000 events.