Culturing and Differentiating Cancer Cell Lines

MDA-MB-231 breast cancer cells^{38 (link)} (HTB-26, ATCC, Manassas, VA), MCF-7 breast cancer cells^{39 (link)} (HTB-22, ATCC), ZR75-1 breast cancer cells^{40 (link)} (CRL-1500, ATCC), PC-3 prostate cancer cells^{41 (link)} (CRL-1435, ATCC), and RWGT2 lung cancer cells^{15 (link),42 (link)} (isolated from bone metastases by Dr. Guise as reported^{15 (link)}) were cultured in Dulbecco's modified Eagle's media (DMEM) (Hyclone, Logan, UT) containing 10% heat-inactivated fetal bovine serum (FBS) (Hyclone). JJN-3 multiple myeloma cells^{43 (link)} (ACC 541, DSMZ) were cultured in RPMI 1640 (Invitrogen, Grand Island, NY) containing 10% heat-inactivated FBS. A549 cancer cells (CCL-185, ATCC) were cultured in 1640 RPMI (Hyclone) containing 10% heat-inactivated FBS. C2C12 myoblast cells (CRL-1772, ATCC) were cultured subconfluently in DMEM containing 10% heat-inactivated FBS. C2C12 myoblasts were differentiated into myotubes by culture in DMEM containing 2% heat-inactivated horse serum (HS) (Hyclone). All cells were maintained at 37° C with 5% CO₂ in a humidified chamber. All cells were verified to be free of mycoplasma contamination via routine PCR testing. No independent verification was completed. Cells treated with recombinant human TGF-β1 (R&D Systems, Minneapolis, MN) were starved in DMEM (no serum) for 16-20 hrs and 5 ng/ml TGF-β1 was added to cells in DMEM.