Poster: High-Content Imaging Detection Of Cellular Lipids To Measure Lipid Toxicity And Cell Differentiation

The detection and understanding of prelethal mechanisms in toxicological profiling and compound screening are important components of drug discovery. Cationic amphiphilic drugs can impact lipid metabolism and inhibit the normal phospholipid metabolism resulting in their intracellular accumulation (phospholipidosis). Other drug classes more adversely affect various aspects of fatty acids or neutral lipid metabolism leading to cytoplasmic accumulation of neutral lipids as lipid droplets (steatosis). We developed fluorescent reagents for performing multiplexed, cellbased high-content imaging cytotoxicity assays to automatically detect and quantify intracellular accumulation of phospho- and neutral lipids in mammalian cell lines. To label phospholipids, live or fixed HepG2 cells were treated with propranolol in a dose-dependent manner with new fluorescent lipid probes with green (ex/ em: 498/532 nm) or orange (ex/em: 557/584 nm) fluorescence emission. Neutral lipid droplets are quantitatively detected either in a steatosis lipid toxicity model in HepG2 cells treated with cyclosporin A or in an adipogenesis model of 3T3L1 fibroblasts differentiated into adipocytes. Neutral lipids were detected with high affinity using a specific probe with red fluorescence emission (ex/em: 656/680 nm) on live or fixed cells without wash steps. The phospho- and neutral lipid probes were multiplexed with each other and other fluorescent markers to detect the lipid compartmentalization in the cell. We found that the cellular lipid detection reagents were specific for their respective targets by their correct cellular location, determined by colocalization analysis with a lysosome marker (for phospholipids) and antiadipophilin immunofluorescence (for neutral lipids). In summary, quantitative imaging analysis was used to explore different lipid toxicity cellular models and lipid accumulation by cell differentiation, was found to be specific and sensitive, and was validated as an accurate screening assay method.