Nile Red Binding to HepG2 Cells: An Improved Assay forIn VitroStudies of Hepatosteatosis

Abstract

Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.

Identification of 5′ AMP-activated Kinase as a Target of Reactive Aldehydes during Chronic Ingestion of High Concentrations of Ethanol

“…Nile Red was used to detect cellular neutral lipid using a modification of previously reported techniques, and Hoechst 33342 was used to measure cellular DNA (32). Cells were collected in 1 ml of phenol red-free, serum-free RPMI 1640, and 50 l of cell suspension was incubated in sterile 96 well fluorescence plates for 1 h at 37°C with 200 l of serum-free, phenol red-free RPMI 1640 containing Nile Red at 20 g/ml and Hoechst 33342 at 10 g/ml.…”

Human SREBP1c Expression in Liver Is Directly Regulated by Peroxisome Proliferator-activated Receptor α (PPARα)

“…Cells were fixed in 4% PFA (pH 7) for 10 min and washed three times with PBS 1ϫ. Nile red staining was performed using the modified protocol described previously (20). Briefly, fixed cells were incubated for 4 h with 1 g/ml of Nile red solution and washed in PBS 1ϫ overnight.…”

Potential Impact of Steatosis on Cytochrome P450 Enzymes of Human Hepatocytes Isolated from Fatty Liver Grafts

“…Long-chain FFA mixture was added to cultures 6 to 8 h after medium renewal. Fat content was determined fluorimetrically by Nile red staining (McMillian et al, 2001). Briefly, hepatocyte monolayers were washed twice with phosphate-buffered saline (PBS) and incubated for 15 min with Nile red solution at a final concentration of 1 mg/ml in PBS at 37°C.…”