Anti ki 67 pe cy7

Cell densities for growth curves of primary B cell derived iGBs were calculated by hemocytometer or using 123count eBeads (Invitrogen). Briefly, 200 μl of cells were mixed with 20 μl of beads and 5 μl propidium iodide (20 μg/ml); 1,000 beads were acquired by flow cytometry. To assess proliferation in vivo, 3 × 10⁶ splenocytes were first surface-stained for GC markers and then treated with fixation/permeabilization solution (cat. #00-5523; eBioscience) for 1 h at 4°C in the dark, washed twice in Perm buffer (eBioscience), followed by 1 h incubation with anti-Ki67-PECY7 (eBioscience) at 4°C and resuspended in PBS + 1% BSA. When necessary, anti-biotin staining was performed following Ki67 staining. Primary B cells in culture were stained with 1 μM CFSE (Invitrogen) on the day of plating, as described in the manufacturer’s protocol, and stimulated with cytokines. CH12 cells were stained with 5 μM CFSE (Invitrogen) 4 d after infection (2 d after puro selection), as described in the manufacturer’s protocol and stimulated with (1 μg/ml rat-antiCD40 [clone 1C10; eBioscience], 10 ng/ml IL-4 and 1 ng/ml TGFβ-1 [R&D Systems]) in the presence of 1 μg/ml puromycin to select for the shRNA vector.