Methionine restriction in cell culture

MDA-MB-231 and MCF10A cells were obtained from American Type Culture Collection (Manassas, VA). MDA-MB-231 cells were maintained in DMEM/F12 media with 10 % fetal bovine serum, and MCF10A cells were maintained in DMEM/F12 media containing 5 % horse serum with the addition of EGF (20 ng/ml), hydrocortisone (0.5 μg/ml), cholera toxin (100 ng/ml), and insulin (10 μg/ml). Cells were routinely passaged weekly and maintained in 5 % CO₂ at 37 °C. Methionine restricted media were specially formulated without methionine, cysteine, and cystine (Life Technologies, Grand Island, NY), and the FBS or horse serum was dialyzed in PBS pH 7.2 three times over a period of 24 h at 4 °C using a Slide-A-Lyzer™ dialysis flask with a 3.5 K MWCO (Thermo Fisher Scientific, Waltham, MA) to remove all amino acids. Media were specially formulated to be 80 % reduced in methionine from the regular DMEM/F12 media (17.24 mg Met/L), with no cysteine or cystine, but with all amino acids and non-essential amino acids (methionine-restricted, cysteine-depleted (MRCD) media). Cells were plated and grown to 80 % confluence, washed once with PBS, and then given either MRCD or regular DMEM/F12 media. Twenty-four hours later, RNA was harvested from cells using Tri-Reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions.