Mycoalert mycoplasma detection kit

Human glioma cell lines U87-MG, U251, U373, T98G and LN229 were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China). Cells were cultured in 10% Dulbecco's modified eagle medium (DMEM, pH = 7.2, Gibco Company, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) in an incubator at 37°C with 5% CO₂. Cell growth and morphology were examined every day and medium was replaced every other day. Serial sub-cultivation was performed when cell density reached 80~85% and subsequent experiments were conducted 12 hours after cell adherence. Doubling time of human glioma cells U87-MG, U251, U373, T98G and LN229 were 32.45 ± 0.52 h, 36.45 ± 0.46 h, 32.45 ± 0.52 h, 36.45 ± 0.46 h and 30.96 ± 0.29 h, respectively. All cell lines were free from mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Sigma-Aldrich Chemical Company, St Louis MO, USA) in the cultivation.

Human breast cancer cell lines, ZR-75-1, MDA-MB-231, MDA-MB-157, MDA-MB-436, MDA-MB-468, CAL51, HCC1954, JIMT-1, MCF-7, T47D, SK-BR-3, BT-474 and HCC1143 and the normal breast cells, MCF12A were obtained from ATCC (Manassas, VA, USA). All the cells were cultured in Dulbecco Modified Eagle Medium (Corning, NY, USA) supplemented with 50 U/ml penicillin/streptomycin, 1% non-essential amino acids and 10% fetal bovine serum (Corning, NY, USA). The media for ER + cell lines were further supplemented with insulin (0.1 µg/ml). The cell lines were authenticated and tested for mycoplasma contamination regularly using MycoAlert mycoplasma detection kit (Sigma, MA, USA). The cumulative culture length of cells between thawing and use in this study was less than 20 passages.