Category: Assay Development and Screening
Nonalcoholic steatohepatitis of NASH is a common liver disease whose major feature is fat in the liver along with inflammation, which can lead to liver damage and cirrhosis. While no definitive cause has been identified, the cellular toxicity of numerous fatty acid metabolites is suspected. The formation of fatty droplets has been proposed as a marker for lipotoxicity caused by fatty acid derived species. HepG2 liver cells exposed to fatty acid mixtures respond in a concentration dependent manner, with over 80% testing positive at high concentrations. While, the liver is the organ primarily responsible for the de novo synthesis of lipids and the conversion of free fatty acids to neutral lipids, in vitro cell types other than liver cells can accumulate neutral lipids. Here we examine the production of lipid droplets in in a number of different cells types, including fibroblasts, epithelial cells and primary hepatocytes, exposed to palmitic and oleic acids
We have shown that liver cells when exposed to various concentrations of a mixture of oleic and palmitic free fatty acids (FFA) accumulate intracellular neutral lipids in a dose dependent fashion up to 1 mM. Dosages of FFA higher than 1 mM result in cell death and the loss of cells. Several other cell lines including HeLa, HT1080 and NIH3T3 cells show a response albeit at different rates and fatty acid concentrations. Following treatment, cells are automatically fixed and stained with Nile red (lipid stain) and DAPI (nuclear stain). Fixed and stained cells were digitally imaged with a Cytation 5 Imaging Multi-mode reader using a 10X objective.
In order to investigate the temporal relationship with lipid droplet formation and fatty acid exposure we have automated the assay process such that challenged cells are fixed and stained at defined intervals over a 24 hour period. Uptake of FFA and their conversion to neutral lipid was monitored over time using a BioSpa robotic incubator to present plates to a liquid handler at periodic times after the addition of FFA in order to stop the reaction by adding fixative to the cells at various times. Automated Image analysis was then used to determine the percentage of cells positive for neutral lipids. Recovery, after removal of the challenging fatty acids, as measured by lipid droplet depletion was also examined in a similar fashion.
We will provide information on lipid droplet formation in a number of cells types. In addition, we describe the automation of the assay workflow using a BioSpa 8 automated incubator system linked to a Cytation 5 Multi-mode imager and a MultiFlo FX Reagent Dispenser and Washer
Peter Banks– Scientific Director, BioTek Instruments, Winooski, VT
Peter Banks is currently the Scientific Director at BioTek Instruments, Inc. His responsibilities include the management of the company’s applications team and providing scientific guidance to the senior management team on new technology and emerging trends impacting the microplate market and its peripheral markets of liquid handling and detection. Before joining BioTek in 2008, Banks was employed in a number of roles within PerkinElmer for a decade. These roles included strategic market analysis, management of PerkinElmer’s BioPharma R&D program and Director of R&D for PerkinElmer BioSignal in Montreal, Canada.