Diacylglycerols (DAG) are key intermediates in the biosynthesis of many cellular lipids and play a fundamental role in biochemical signaling. Cell Biolabs’ DAG Assay Kit simple 96-well fluorometric assay that measures diacylglycerol content in samples by a coupled enzymatic reaction system. First, kinase is used to phosphorylate DAG samples, yielding phosphatidic acid. Next, a lipase is used to hydrolyze phosphatidic acid to glycerol-3-phosphate. Finally, the glycerol-3-phosphate product is oxidized by glycerol-3-phosphate oxidase (GPO), producing hydrogen peroxide which reacts with the kit’s fluorometric probe. The kit contains a DAG Standard and has a detection sensitivity limit of 15 µM. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, standards and unknown samples. Note each sample replicate requires 2 assays, one of which is a negative control to be subtracted as background.