Pe conjugated anti mouse iga

For CSR analysis, CH12 cells were stained with PE-conjugated anti-mouse IgA (Southern Biotech), and ex vivo mouse B cells were stained with PE-conjugated anti-mouse IgG1 clone A85-1 (BD Biosciences). For BrdU-PI assays, cells were incubated with 10 μM BrdU for 1 hour followed by ethanol fixation for 16 hours. Cells were then washed and incubated in 2 M HCl for 30 minutes, washed and stained with FITC-conjugated anti-BrdU clone PRB-1 (eBiosciences), and then washed and incubated in 20 μg/mL PI and 10 μg/mL RNase A for 30 minutes. For apoptosis analysis, CH12 cells were stained for Annexin V using the Annexin V-APC Apoptosis Detection Kit (eBiosciences). For mitotic index analysis, cells were CIT stimulated or irradiated and stained 24 hours post-stimulation with Alexa Fluor 488 conjugated anti-phospho-histone H3 and 20 μg/mL PI. For the above assays, cells were acquired with a FACSCalibur (BD Biosciences) and analysis performed with FlowJo VX (Tree Star Inc). For CFSE analysis, cells were pulsed with 5 μM CFSE (Celltrace, Life Technologies), washed, and CIT stimulated, as described above. Cells were collected 0, 12, 24, 36, and 48 hours following CFSE pulse and stimulation, stained, and acquired with an LSR II flow cytometer (BD Biosciences) and analyzed.