Extracellular Acidification Rate (ECAR) Fluorometric Assay Kit

① Pre-warm Reagent 1 and the probe working solution to 37°C. Set the detection instrument to 37°C in advance; pre-warming for 1 hour is recommended. ② ECAR readings correlate with cell type and density. For preliminary experiments, use high cell seeding densities. If fluorescence changes per unit time are minimal, adjust cell density accordingly. ③ After adding the probe working solution to cell samples, avoid incubating them at 37°C in a 5% CO? cell culture incubator to prevent high CO? concentrations from entering the detection system and affecting results. ④ If drug treatment is required for cell samples and the drug exposure time exceeds 1 hour, it is recommended to perform drug treatment prior to seeding. ⑤ Reagent 1 contains glucose sufficient to support short-term cellular metabolic activity. During cellular metabolism, energy is primarily supplied by aerobic or anaerobic respiration. When cells undergo anaerobic respiration, large amounts of acidic substances are produced, leading to acidification of the extracellular space after pumped outer space. Therefore, the extracellular acidification rate (ECAR) serves as a crucial indicator for assessing the acidic substances generated by anaerobic respiration during cellular metabolism. Changes in ECAR reflect alterations in cellular metabolic status. This assay kit employs a specific probe (Ex/Em: 490 nm/535 nm). When the pH of the extracellular environment changes, the fluorescence intensity of the probe also changes accordingly. An acidified extracellular environment causes a decrease in the probe's fluorescence intensity, thereby enabling the detection of the extracellular acidification rate.