Nile red binding to HepG2 cells: An improved assay for in vitro studies of hepatosteatosis

Michael K. McMillian, Elfrida R. Grant, Zhong Zhong, J. Brandon Parker, Li Li, Robert A. Zivin, Michael E. Burczynski, Mark D. Johnson

Research output: Contribution to journalArticlepeer-review

92 Scopus citations


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 as say 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 μM 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.

Original languageEnglish (US)
Pages (from-to)177-190
Number of pages14
JournalIn Vitro and Molecular Toxicology: Journal of Basic and Applied Research
Issue number3
StatePublished - 2001
Externally publishedYes

ASJC Scopus subject areas

  • Toxicology
  • Health, Toxicology and Mutagenesis


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