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Modeling Nonalcoholic Steatohepatitis (NASH): Automated Assay for Lipid Accumulation in Liver CellsDownload
February 13, 2017
Authors: Xavier Amouretti and Paul Held; BioTek Instruments, Inc.
Nonalcoholic steatohepatitis, or NASH, is a common, often “silent” liver disease that resembles alcoholic liver disease, but occurs in people who drink little or no alcohol. The etiology of the disease is the accumulation of neutral lipid droplets within the liver cell, which can be tracked with Nile red staining. To date, research in this area has suffered from the lack of a suitable human cell model as well as the means to automate the assay process and subsequent microscopic image analysis.
We have shown here that liver cells, when exposed to various concentrations of a mixture of oleic and palmitic free fatty acids (FFA), accumulated intracellular neutral lipids in a dose dependent fashion up to 1 mM FFA treatment. Doses of FFA higher than 1 mM resulted in cell death and the loss of cells. Following treatment, cells were fixed and stained with Nile red (lipid stain) and DAPI (nuclear stain) using automation. Fixed and stained cells were digitally imaged with a Cytation™ 5 Imaging Multi-Mode Reader using a 10x or 20x objective. Uptake of FFA and conversion to neutral lipid droplets was monitored over time using a BioSpa™ 8 Automated Incubator to present plates to a MultiFlo™ FX Multi-Mode Dispenser at periodic times after the addition of FFA in order to stop the reaction by adding fixative to the cells. Automated Image analysis was then used to determine the percentage of cells positive for neutral lipids.
Cytation™ 5 Imaging Multi-Mode Reader