Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual-emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the National Institutes of Health library. The screening data from the plate reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate reader assay is a powerful high-throughout screening platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.
Keywords: 1536-well high-throughput screening; H9c2 cardiomyocytes; Nile red fluorescence; high-content analysis; human primary myocytes; lipid accumulation; phenotypic screening.