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Cells were grown in a serum-free mammosphere culture medium (MammoCult, StemCell Technologies) supplemented with MammoCult Proliferation Supplements.
293T cells were cultured in DMEM (Life technologies) supplemented with 10% FCS (Greiner bio-one), 2 mM L-glutamine, 100 units.ml−1 penicillin and 0.1 mg.ml−1 streptomycin (all Gibco).
After treatment, mTeSR™1 medium (StemCell Technologies) supplemented with 2 mM CaCl2 was added to the cells and the cells were transferred to Matrigel (BD Biosciences -coated Biosciences -coated for 2–4 h.
Human CB CD34+ cells were pre-stimulated for 48 h with StemSpan (StemCell technologies) supplemented with 300 ng/ml hSCF (Peprotech), 100 ng/ml hTPO (R&DSystems, Minneapolis, MN) and 100 ng/ml Flt3-L (Invitrogen, Carlsbad, CA).
After extensive washing to remove residual maternal blood, three grams of placental fragments were cultured in 20ml of culture medium made up of RPMI 1640 (Gibco BRL life technologies) supplemented with 10% fetal bovine serum, 1% L-glutamine (Gibco BRL life technologies), and 1% penicillin/streptomycin (Gibco BRL life technologies).
C2C12 myogenic cell lines were cultured in DMEM (GIBCO-Life Technologies) supplemented with 10% fetal bovine serum.
Cells were harvested directly on tissue culture plates with 1% triton cell lysis buffer (Cell Signaling Technologies) supplemented with protease and phosphatase inhibitors.
In both cases, 10 cells/2 ml were cultivated in Mammocult Basal medium and 10% proliferation supplement (StemCell Technologies), supplemented with 0.2% heparin, and 10−3 M hydrocortisone on nonadherent plates at 37°C, 5% CO2.
Pancreatic cancer cell lines ASPC1 and BxPC3 were purchased from the ATCC and cultured in RPMI-1640 medium (31870; Gibco-Life Technologies) supplemented with 5 mM L-glutamine (25030; Gibco-Life Technologies) and 10% FCS Gold (PAA Laboratories GmbH, Colbe, Germany).
All cells were cultured in RPMI GlutaMAX medium (Gibco/Life Technologies) supplemented with penicillin, streptomycin and 10% (vol/vol) GemCell human serum AB (Gemini Bio-Products, West Sacramento, CA, USA).
