The following human TNBC cell lines were used: BT549 and MDA-MB-468 (both obtained from A. Schulze, Biocenter, Wurzburg, Germany, and originally purchased from the American Type Culture Collection, VA, USA) and CAL-120 (DSMZ, Braunschweig, Germany). All cell lines were authenticated using the Promega GenePrint 10 Kit (Promega, WI, USA) and tested negative for mycoplasma infection using the MycoAlert Mycoplasma Detection Kit (Lonza, Bazel, Switzerland). Before performing experiments to compare different media, cells were maintained in DMEM-F12 (cat. no. 21331046; Thermo Fisher Scientific) supplemented with 2 mM glutamine (Thermo Fisher Scientific) and 10% FBS (Thermo Fisher Scientific). Cells were cultured at 37°C and 5% CO2. To determine the doubling time, MDA-MB-468 cells were cultured in DMEM-F12 and Plasmax supplemented with 2.5% FBS for at least 10 consecutive passages. At each passage, the cells were trypsinized and counted, and the same number of cells (500,000 or 1,000,000) were seeded in a 10-cm dish with the respective media and allowed to grow until 80% confluent. For the identification of the Plasmax component responsible for HIF1α stabilization, BT549 cells were preincubated in DMEM-F12 or Plasmax (both supplemented with 2.5% FBS) for 2 days and subsequently (day 2) seeded in six-well plates at 50,000 cells per well in 2 ml of the respective culture medium. After 24 hours (day 3), culture medium was changed to 8 ml of medium (DMEM-F12, DMEM, or RPMI 1640 for cells preincubated in DMEM-F12; Plasmax for cells preincubated in Plasmax) with different concentrations of pyruvate. Cells were allowed to proliferate for 72 hours, after which cell lysates were prepared (day 6) for immunoblotting.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.