4.17. Measurement of Extracellular Lactate

Using the Lactate-Glo™ assay kit (Promega, VWR), the lactate levels exported by A375 and SK-MEL-5 cells to the culture medium were determined according to the manufacturer’s instructions. Briefly, 1.2 × 10⁵ A375 cells/well and 1.5 × 10⁵ SK-MEL-5 cells/well were seeded in six-well plates, followed by treatment with a concentration range of SLMP53-2. After 8 h treatment, the culture medium was collected and diluted in phosphate saline buffer (1:200). Then, 50 μL was transferred to a 96-well assay plate and 50 μL of Lactate Detection Reagent Mix was added. The plate was kept 60 min at room temperature followed by luminescence assessment using the Bio-Tek Synergy HT plate reader (Izasa).