²H content decreases generation after generation among cells that proliferate in absence of ²H₂O. (A) PBMCs from CLL 355 at the third time point during ²H₂O protocol (Table 2) were loaded with CFSE and cultured with CpG 2006-G5 (5 μg/mL), IL-2 (5 ng/mL), and IL-15 (5 ng/mL) to induce proliferation. Cells were harvested after 5 days and stained with annexin V–APC to exclude dying/dead cells. (B) Annexin V⁻ cells were analyzed for CFSE content and 3 generations of cells were flow-sorted based on the indicated gates (0, 1, 2). Gate 0 represents undivided cells. Note that whereas gates 0 and 1 contained discreet CFSE peaks, gate 2 probably contained a main peak and a smaller one (dotted lines), possibly representing another generation of progeny. (C) A decreased fraction (f) of labeled cells was found generation after generation.