B cll cell isolation kit

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. CD19+ CD5+ B-cells were isolated from PBMCs using magnetic-bead-activated cell sorting, using a B-CLL cell isolation kit (Miltenyi Biotec). To evaluate the effect of AA on normal lymphocytes, naïve B-cells were isolated from donor lymphocyte infusions using a Naïve B-cell Isolation kit (Miltenyi Biotech). The purity assessed by CD19 expression on flow cytometry was around 98%. OSU-CLL cells were a gift from E. Hertlein and colleagues [28 (link)]. JVM3 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Freshly isolated B-cells and CLL cell lines were cultured in RPMI 1640 medium (PAN Biotech, #P04–16500) with 10% fetal bovine serum (FBS) (PAN Biotech, #P30–3306) and L-glutamine, penicillin and streptomycin (1%) (Eurobio Scientific). When indicated, CLL B-cells were cultured in Iscove’s modified Dulbecco’s Medium (IMDM) (Merck, #FG0465) and alpha-MEM medium (Sigma Aldrich, #M4526) (n = 7). Cells were cultured at a density of 4 × 10⁵/ml in 48-well plates and were treated with either vehicle or AA or drugs for 24 h. To simulate hypoxia condition, cells were cultured in presence of 100 μM Cobalt(II) chloride hexahydrate (CoCl₂.6H₂O, Sigma Aldrich) for 24 h. Cells were then washed and incubated with AA for 24 h.

SKW3-T18 cells were cultured in complete RPMI-1640. Cell line identity was confirmed using short tandem repeat analysis (Powerplex 16 System, Promega, Southampton, UK) and absence of mycoplasma was confirmed using Mycoplasma PCR detection kit (Applied Biological Materials, Richmond, Canada). CLL cells were purified from PBMCs using a B-CLL cell isolation kit (Miltenyi Biotec) and incubated with soluble antibody or antibody-coated beads (2:1 bead:cell ratio) for 15 or 60 min, respectively. CLL cells were then co-cultured with SKW3-T18 cells at a final density of 100,000 SKW3-T18 and 400,000 CLL cells in a total volume of 0.2 mL. Supernatants were collected after 24 h and IL-2 quantified using an enzyme-linked immunosorbent assay (ELISA; R&D Systems) according to the manufacturer’s instructions. All assays were performed in duplicate.