Culturing Murine TNBC Cell Lines

Firefly luciferase-expressing 4T1 mammary tumor cells that resemble metastatic human TNBC were provided as a kind gift from Prof. Clare Isacke (Breakthrough Breast Cancer Research Center, London, UK) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (both from Sigma-Aldrich, Overijse, Belgium) at 37°C and 5% CO₂. Py230 mammary tumor cells, derived from the American Type Culture Collection (ATCC) and reported to model TNBC,^{18 (link),20 (link)} were cultured in Ham’s F-12 K Medium (Thermo Fisher Scientific) supplemented with 5% heat-inactivated FBS (Thermo Fisher Scientific), 0.1% MITO+ Serum Extender (Corning, New York, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C and 5% CO₂. Checks for mycoplasma and other bacterial contamination were routinely performed using a PlasmoTest^TM mycoplasma detection kit (Invivogen, San Diego, USA). Harvesting of the cells involved washing with phosphate buffered saline (PBS) and incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) for 5 minutes (min). The cells were subsequently centrifuged at 805 × g for 5 min, resuspended in PBS and counted with a Bürker chamber.