Isolation and Culture of Primary CLL Cells

All CLL patients provided written informed consent according to the Declaration of Helsinki to the University of Oklahoma Health Sciences Center (OUHSC) Institutional Review Board, which approved these studies. Primary CLL cells were isolated and purified from blood of previously untreated CLL patients at or near diagnosis (n = 19; clinical features are shown in Supplementary Table 1) using RosetteSep B-cell enrichment kit (STEMCELL Technologies). CLL patients were chosen randomly independent of their prognostic factors however, previously treated patients were excluded from the study. The typical purification range of CD5⁺/CD19⁺ CLL cells for this work was >99%. Purified normal CD19⁺ peripheral B-cells (purification range: >95%–99%) from healthy, age-matched individuals (n = 8) were purified as described earlier [17 (link)] and included as controls wherever appropriate. Cells were cultured in serum-free AIM-V (GIBCO) medium as needed. Of note, we did not supplement fetal bovine serum (FBS) to CLL cell cultures as prior study found that FBS induces spontaneous apoptosis in CLL cells [28 (link)]; instead, we used serum-free AIM-V basal media that contain human serum albumin to support primary CLL cell growth [29 (link)].