Lymphocyte cell separation medium

The study population consisted of 108 B-CLL patients. Peripheral blood samples were collected in heparin-coated tubes from all B-CLL patients following informed consent, in accordance with the Declaration of Helsinki and in agreement with institutional guidelines (University-Hospital of Ferrara). The main demographic and clinical parameters of the patients were abstracted from clinical records. B-CLL samples were also characterized by CD38 surface expression, interphase FISH and IgVH status (Table 1). All patients had been without prior therapy at least for three weeks before blood collection. Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane Laboratories, Hornby, ON). T lymphocytes, NK lymphocytes, granulocytes and monocytes were negatively depleted from peripheral blood leucocytes (PBL) with immunomagnetic microbeads (MACS microbeads, Miltenyi Biotech, Auburn, CA) and purity (> 93%) of resulting CD19⁺ B-CLL population was assessed by flow cytometry as previously described [24 (link)]. Viability of the cells was analyzed at the end of the purification procedure by Trypan blue dye exclusion as previously described [43 (link)].

Tumors were harvested from mice and minced into small pieces, followed by a one-hour incubation with RPMI supplemented with 1% FBS, 2 mg/mL Collagenase Type I (Worthington Biochemical Corporation) and Collagenase Type IV (Worthington Biochemical Corporation), and 30 mg/mL DNase (Sigma-Aldrich). After incubation, tumor samples were mashed against a 70 μm cell strainer to harvest immune cells, which were subsequently enriched by Lymphocyte Cell Separation Medium (Cedarlane Labs) and lysed in ACK lysis buffer (Lonza). Single-cell suspensions were then used for flow cytometry staining and fluorescence-activated cell sorting (FACS).