Anti mouse cd 19 fitc

For flow cytometry, 2 μL of the mixture of 2–2 antibodies was added to 50 μL of whole blood in a tube. Anti-Mouse CD4-PE (BD Pharmingen, 553048) / Anti-Mouse CD 19-FITC (eBioscience, 11-0193-82) or Anti-Mouse CD8a-PE (BD Pharmingen, 553032) / Anti-Mouse F4/80-Alexa Fluor 647 (Caltag, MF48021) were used together. Samples were incubated for 30 min in the dark at room temperature (RT). Then, 1 mL of 1 × eBioscience 1-step Fix/Lyse solution (eBioscience, 00-5333-57 10 × Solution) was added to the samples and vortexed gently. Samples were incubated for 30 min in the dark at room temperature. After a washing step (centrifugation at 500 × g for 5 min, wash once with 1 ml flow stain buffer, and spin again), cells were resuspended in 200 μL flow stain buffer. Samples were analyzed by a flow cytofluorometer (BD FACSCalibur). Data were analyzed using FlowJo software: FSC/SSC panel: R1 gate: living cells; FL1/FL2 panel: CD19-FITC/CD4-PE, CD19- and CD4-positive cells in R1; FL2/FL4 panel: CD8-PE/F4/80-A647, CD8- and F4/80-positive cells in R1.

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).