Figure 4.

CTV and CTY‐labeling of human naïve and memory B cells do not impact cellular behaviors. Human splenic naïve and memory B cells were labeled with CTV or CTY as indicated and cultured in the presence of CD40L (100 ng mL⁻¹) and IL‐21 (50 ng mL⁻¹). After 4 days, cells were harvested, washed and re‐stimulated under the same conditions and (a) the gMFI of CTV and CTY assessed after 24 h of re‐culture, then daily from days 4–10. (b) The number of live cells present at each time point was assessed in cultures of human naïve (left) and memory (right) B cells. Mean live cell numbers ± SEM of replicate wells are shown. No significant differences were observed between the live cell number of unlabeled cells or cells labeled with each division tracking dye (Kruskal–Wallis test with Dunn's multiple comparisons, GraphPad Prism). (c) The mean division number of dividing cells was assessed in cultures of activated naïve (left) and memory (right) B cells. No significant differences were observed between the mean division number of unlabeled cells or cells labeled with each division tracking dye (One‐Way ANOVA, GraphPad Prism). Data are representative of (A) two (CTV) and at least 5 (CTY) independent experiments using cells from different donors and (B) and (C) 3 independent experiments. CTY, CellTrace Yellow; CTV, CellTrace Violet; CTFR, CellTrace Far Red; gMFI, geometric mean fluorescence intensity.