Recursos - Pôsteres Científicos
Monitoring Cellular Stress Response and Inhibition of GPCR-Dependent Calcium Signaling Using Expressed BiosensorsDownload
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December 09, 2018
Endoplasmic reticulum (ER) mediated cell stress and the unfolded protein response (UPR) serve as a major stress pathway in eukaryotic cells. ER stress and the UPR can be triggered by a number of cellular perturbations including misregulation of ER Ca2+, accumulation of misfolded proteins within the lumen of the ER, and reactive oxygen species (Grootjans et al., 2016; Scheper and Hoozemans, 2015; Zeeshan et al., 2016). ER stress has been implicated in a wide range of diseases including neurodegenerative diseases, cancers, diabetes and ischemia (Auf et al., 2010; Kammoun et al., 2009; Kudo et al., 2007; Scheper and Hoozemans, 2015), and is also an area of emerging interest for drug development and toxicity testing (Huang et al., 2016; Kim et al., 2017; Plate et al., 2016; Roth et al., 2014). Activation of proteostatic pathways, such as the UPR and ER stress serves as an early indicator of cell stress or toxicity, which is a useful mechanism to detect drug or compound induced cell stress (Deavall et al., 2012; Foufelle and Fromenty, 2016).
The UPR consists of three arms, each activating a unique mechanism to regulate the cellular response to ER stress (Grootjans et al., 2016). Methods to detect activation of ER stress and the UPR range from PCR based assays to fluorescent indicators (Iwawaki et al., 2004; Rong et al., 2015). However, few current detectors of ER stress are optimized for detecting both chemical as well as genetic induced stress, and are irreversible indicators. Here we demonstrate the use of a new and commercially available ER stress sensor developed by Montana Molecular to detect chemically induced cell stress by thapsigargin, a potent SERCA pump inhibitor, and simultaneously detect its effects on Gq mediated cell signaling (Figure 1). Importantly, high content analysis of ER stress using the BioTek Lionheart™ FX allows the detection of both the onset and recovery from thapsigargin induced cell stress as well as simultaneous detection of Gq mediate cell signaling over a 24 hour period. In sum, combining live cell detection of cell stress and signaling with kinetic high content monitoring allows precise detection of changes in cellular stress levels and its implications on cellular signaling events.
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