Human MM cell lines U266, RPMI8226 and OPM-2 were purchased from DMSZ Germany, whereas MM1.S cells were kindly provided by Dr. Renate Burger (Division of Stem Cell Transplantation and Immunotherapy, Kiel, Germany) and cultured in RPMI-1640 media (BioWhittaker, Walkersville, MD, USA) supplemented with 10% of FBS (BIOCHROMAG, Leonornstr., Berlin, Germany), 2 mM L-Glutamine (BIOCHROMAG, Leonornstr., Berlin, Germany), streptomycin 100 U/ml, penicillin 100 U/ml and maintained in 5% CO2 at 37°C. Hypoxia (1% pO2) was generated in an Vivo2300 hypoxic workstation (Ruskinn Technologies, Ireland). Mononuclear cells from bone marrow aspirates or peripheral blood samples were isolated by density gradient centrifugation over Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ, USA). CD138+ and CD34+ cells were then isolated by immunomagnetic bead positive selection in a Mini MACS LS column following the manufacturer's protocol (Milteny Biotech, Auburn, CA). The purity of MM cells was confirmed by flow cytometry analysis using phycoerytrin-conjugated anti CD138/CD34 antibody (Milteny Biotech, Auburn, CA). Patient samples were collected after informed consent.
To analyze S-phase progression MM cells were synchronized in the presence of 2 mM Thymidine (Sigma Aldrich, St Louis, MO, USA) for 48h, then released in complete media for up to 48h. Cell cycle analysis was then performed.
To analyze S-phase progression MM cells were synchronized in the presence of 2 mM Thymidine (Sigma Aldrich, St Louis, MO, USA) for 48h, then released in complete media for up to 48h. Cell cycle analysis was then performed.