Cell Viability Assay with Metabolites

To analyze the cell viability in response to the metabolites, a resazurin-based assay with the reagent PrestoBlue (ThermoFisher; Walthman, MA, USA) was used (19 (link)). The assay was performed according to the manufacture's protocol. Briefly, cells were seeded with a density of 10,000 cells/well in 96-well-plates (Corning 3610; Corning, NY, USA). Cells were treated with different concentrations of the metabolites (1 μM−1 mM) for 24 h at 37°C (with 5% CO₂). After the treatment, cells were incubated with media containing 1× PrestoBlue for 1 h at 37°C (with 5% CO₂) and fluorescence intensity was measured at Ex/Em 560/590 nm (Tecan infinite M200; Tecan Group Ltd., Männedorf, Switzerland).