Ccrf cem

Human pre-B ALL cell lines SEMK2, RS4;11, 697 and a T-ALL cell line CCRF-CEM were obtained from DSMZ (Braunschweig, Germany). Cell lines were maintained at a density of 0.6–2.0×10⁶/ml in RPMI-1640 medium (Lonza) supplemented with 10% heat inactivated fetal bovine serum (Biovest), 2 mM L-glutamine, 10 mM HEPES and 100 U/ml of penicillin/streptomycin (Lonza). Peripheral blasts from adult pre-B-ALL patients were isolated using density gradient centrifugation (Ficoll-Hypaque; density 1.077 g/mL) within 24 hours from harvesting. If necessary, ALL samples were further purified to achieve more than 90% blasts by removing nonmalignant cells using immunomagnetic beads (B-cell isolation kit, Miltenyi). Cells were then frozen in liquid nitrogen or cultured at 2×10⁶/ml concentration in RPMI-1640 medium (Lonza) containing 20% heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES and 100U/mL of penicillin/streptomycin. The study was conducted after approval of Bioethics Committee (Komisja Bioetyczna, http://www.ihit.waw.pl/komisja-bioetyczna.html). The study was conducted according to Declaration of Helsinki principles. Patients provided written informed consent, and all sensitive information were blinded and data were analyzed anonymously.