Rpmi 1640 media

We used an experimentally evolved cancer cell line derived from a non-small cell lung cancer cell line obtained from the American Type Culture Collection – ATCC (Manassas, VA, USA). This original cell line (NCI-H2122) was established from the pleural effusion of a 46-year-old female with stage-4 adenocarcinoma and grows as a mixture of two distinct phenotypes: clusters of cells growing in suspension and adherent cells that grow as a monolayer. By selectively passaging only the adherent or suspension cell populations, two cell lines were experimentally evolved: one that grows as cell clusters in suspension (referred to as the H2122 Suspension-Selected or SS line) and one that grows as adherent cells [45 (link)]. The SS line has been previously shown to be a viable model-system for CTC clusters [44 (link)]. The SS cells were grown at 37 °C and 5% CO₂ in RPMI-1640 media (MP Biomedicals) supplemented with 10% FBS and 1% Penicillin/Streptomycin mix. The concentrations of glucose and glutamine in the media were adjusted to 5 mM and 0.5 mM respectively, to simulate physiological levels. Cells were passaged every 2–3 days at a 1:3, 1:4 or 1:5 ratio depending on the required cell density.

For the genotoxicity studies, positive controls were acquired from Sigma Aldrich (2-aminoanthracene, 9-aminoacridine, sodium azide, and cyclophosphamide monohydrate), Tokyo Chemical Industry (2-nitrofluorene), and MP Biomedicals (mitomycin C). Fetal bovine serum (FBS) was procured from Gibco. RPMI-1640 media was procured from MP Biomedicals. Salmonella strains were procured from Moltox, USA, and S9 fractions were prepared fresh, in-house.