A low-cost, enzyme-coupled fluorescent assay for rapid quantification of glycolysis rate of cells

Glycolysis is the predominant energy-yielding metabolic pathway in most cancer cells and rapidly proliferating cells. Currently available methods for glycolysis rate analysis are either time-consuming or cost-intensive/specialized equipment-dependent. The present study demonstrates a convenient, fast, and low-cost enzyme-coupled fluorometric assay for rapid quantification of glycolysis rate in small amount of cells. This assay involves the oxidation of cell-secreted lactate to produce hydrogen peroxide (H₂O₂) and subsequent conversion of Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) to fluorometric resorufin, in the presence of lactate oxidase (LOx) and peroxidase. High detection sensitivity and stability were realized by optimization of assay medium composition, enzyme and substrate concentration, and assay procedure. The lower limit of detection on HeLa cells was achieved on 50 cells per sample and the optimized linear range of the detection was 250-7000 cells per sample (r² = 0.9842). The repetitive intraday and interday measurements of HeLa cell provided small variance and were highly agreeable with the results of endpoint method, which is a conventional validated method but detects lactate in relatively long time of larger cell population. The present assay was successfully applied on measuring the glycolytic parameters of human cancer cells (HeLa, HepG2) and mouse immune cells (T cells, macrophages), indicating great potential for wide application in cancerous and immunological research.