Rosettesep b cell enrichment kit

The tumor load of CLL PB samples ranged from 51 to 100%. Purified CLL cell samples (n = 46) were prepared with negative selection of CD19⁺ B cells from whole blood using the RosetteSep B-cell enrichment kit (StemCell Technologies, Vancouver, BC, Canada) following the manufacturer’s instructions.

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)].

As input material, B lymphocytes separated from peripheral blood using gradient centrifugation on Ficoll-Pague PLUS (GE Healthcare, Uppsala, Sweden) coupled with the RosetteSep^® B Cell Enrichment Kit (StemCell Technologies Inc., Vancouver, Canada) were used. RNA was isolated with TRI Reagent (MRC, Cincinnati, USA). As positive controls, RNA samples from cell lines NALM6 and MEC-1 were used. Then, RNA was reverse transcribed into cDNA using SuperScript^® II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Gene expression (APEX1, CDK4, CDKN1A, CDKN1B, CDKN2B, DUSP1, GADD45A, NCL TERT) was analyzed by real-time PCR on the QuantStudio™ 12K Flex system (both Thermo Fisher Scientific, Waltham, USA) using ThermoFisher Scientific TaqMan assays. The HPRT1 and TBP genes were used as endogenous controls. All reactions were pipetted in triplicates. After removing outlying Ct values (i.e., the values differing from the remaining two replicates by ≥0.3 Ct; 4.6% of Ct values) to correct on the technical accuracy of the method, relative quantification using the 2^-ΔΔCT method was performed.