Material And Methods : Bm Mscs Cultivation

859 WordsDec 3, 20164 Pages
Material and Methods: BM-MSCs cultivation: BM-MSCs were obtained from Institute Pasture of Iran and were cultured in Low Glucose Dulbecco 's modified Eagle 's medium (Low Glc-DMEM) (Gibco Invitrogen, Karlsruhe, Germany) with 10% FBS (Gibco Invitrogen, Karlsruhe, Germany) and 1% penicillin and streptomycin (Sigma-Aldrich, Schnelldorf, Germany). Then, BM-MSCs were incubated in humified incubator at 37˚C and 5% CO2. Cell’s medium was replaced every two days. U266 cell line: Confirmed U266 cells, a human myeloma cell line, were purchased from Institute Pasture of Iran. U266 cells were grown in RPMI-1640 (Gibco Invitrogen, Karlsruhe, Germany) with 10% FBS (Gibco Invitrogen, Karlsruhe, Germany) and 1% penicillin and streptomycin (Sigma-Aldrich, Schnelldorf, Germany) in T25 flask culture (Orange Scientific, Braine-l 'Alleud, Belgium). Then, U266 cells incubated in humified incubator at 37˚C and 5% CO2. The medium was changed every day during all steps of experiments and Trypan blue staining was used for viability analyzing. BM-MSCs derived C.M preparation: The BM-MSCs were cultured in T25 flask culture (Orange Scientific, Braine-l 'Alleud, Belgium) with DMEM medium and incubated for reaching to 60-70% confluency. In the next step, DMEM medium was replaced with RPMI-1640 medium without FBS and was incubated for 72 hours (hrs). After incubation; medium was collected, cells and debris were removed by centrifugation. Supernatant was concentrated by 10 kDa MW cut-off ultrafiltration

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