Rat igg1 apc

Single-cell suspensions of splenocytes and lymph node cells were treated with RBC lysis buffer (Sigma), and washed with FACS buffer (PBS plus 1% FBS). These cells were incubated with specific surface-binding antibodies for 30 min at 4°C. Antibodies included anti-mouse CD3e-percp-cy5.5, anti-mouse CD19-FITC, anti-mouse CD44-FITC, anti-mouse CD62L-PE, anti-mouse B220-APC, anti-mouse CD80-APC, anti-mouse CD86-APC and rat IgG₁-APC isotrol control and rat IgG_2a-PE isotrol control (all from eBioscience, San Diego, CA), anti-mouse CD138-PE (Miltenyi Biotech, Bergisch Gladbach, Germany), anti-mouse CD4-APC, anti-mouse CD8-APC (BD pharmingen). In some experiments, splenocytes were stimulated with LPS (1 μg/ml) or PBS for 24 h before stained with fluorochome-conjugated CD80, CD86 and CD19 antibodies. For JC-1 staining, splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies (1 μg/ml for each, BD pharmingen) for 24 h and then incubated with 5 μg/ml JC-1 (Biotium, Hayward, CA) for 30 min at 37°C. Samples were analyzed by flow cytometry on a FACScan flow cytometer (Becton Dickinson).

Mice received 1 mg BrdU (Sigma-Aldrich) i.p. daily for the final 4 days before harvest of pinnae in order to determine in vivo cell proliferation. DEC were then recovered, and blocked with 1 μg anti-CD16/32 mAb in goat-serum. Subsequently, DEC were labelled for surface expression of CD3, CD4, MHC-II, F4/80 and CD11b (all eBioscience) in PBS supplemented with 1% FCS. Cells were then washed and incubated in 1x Fixation/Permeabilisation buffer (eBioscience) for 1 hour at 4°C before being washed and incubated for 1 hour at 37°C in 100 μg DNase (Sigma-Aldrich). Finally, cells were labelled for 45 minutes at room temperature with anti-BrdU APC mAb, or rat IgG1 APC (eBioscience), in 1x Permeabilisation buffer as per the manufacturer’s protocol.