Kinetics of and requirements for the generation of vesicles derived from cultured GC B cells. GC B cells were stained with PKH26-GL, cultured for 2 hours, and the cell-free SN was analyzed by flow cytometry. (A) A dot plot of SS versus FL2 parameters was used to define GC B-cell–derived membranous particles. (B) The count of PKH26-GL⁺ particles detected in the SN obtained for indicated times was recorded. Results of one experiment representative of three are shown. (C) PKH26-GL–stained GC B cells were cultured for 12 hours at 4°C and at 37°C in the absence (control culture) and in the presence of cycloheximide (Cx, 10 μg/mL), cytochalasin B (CkB, 5 μg/mL), EDTA (1 nmol/L), anti-CD40 MoAb (1 μg/mL) + IL-4 (2 ng/mL), PMA (10 ng/mL), and VAD-fmk (200 μmol/L). Values of the counts of PKH26-GL⁺ particles were recorded and expressed as percentages of the control figures. Results represent the mean ± SEM of four experiments.