Ficoll density gradient centrifugation

BM samples were collected for routine diagnostic or evaluation purposes after patients' informed consent and in accordance with the Declaration of Helsinki. All research was approved by the local ethical committee (B.U.N. 143201316382). BM mononuclear cells were obtained after Ficoll density gradient centrifugation (Nycomed, Lucron Bioproducts, Zurich, Switserland) and purified using MACS Cell Separation MS Columns (Miltenyi Biotec, Bergisch-Gladbach, Germany) and CD138 microbeads according to the manufacturer's protocol. Purity of the samples was evaluated by flow cytometry (FACS Canto, BD Biosciences, Franklin Lakes, USA) and analysed using FACSDiva software (BD Biosciences).

BM samples were collected from MM patients for diagnosis and research purpose and blood was collected from healthy individuals after written informed consent in accordance with the Declaration of Helsinki. The research is approved by the Ethic Board of UZ Brussel (B.U.N. 143201316382) and the Tumourbank of Lille (CSTMT102). Mononuclear cells were obtained after Ficoll density gradient centrifugation (Nycomed, Lucron Bioproducts, Zurich, Switzerland) and purified as previously described [57 (link)]. Primary BMSCs were obtained from healthy subjects, and maintained in DMEM medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (HyClone, USA), 10% horse serum (Invitrogen, USA), 100 U/mL penicillin/streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Peripheral blood mononuclear cells were isolated from heparinized blood of CLL patients by Ficoll density-gradient centrifugation (Nycomed, Oslo, Norway). Monocytes were removed by plastic adherence, and T cells by sheep erythrocyte rosetting. All CLL samples contained more than 96% CD19⁺/CD5⁺ CLL cells, as assessed by flow cytometry (EPICS-XL-MCL; Beckman Coulter, Fullerton, CA).