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Imaging-based Cytotoxicity AssaysTélécharger
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February 05, 2018
Authors: Sarah Beckman and Brad Larson, BioTek Instruments, Inc., Winooski, VT USA
Cell death occurs throughout the life of an organism, and this is critical for developmental plasticity and organismal health, in part by eliminating unneeded and unhealthy cells in a timely and effective manner. However, dysfunctional programmed cell death leads to diseases such as cancer, neurodegeneration, and ischemic damage. Two classic pathways of cell death are apoptosis and necrosis. Apoptosis is an active, programmed process of autonomous cellular dismantling that avoids eliciting inflammation. Necrosis is a more passive process with uncontrolled release of inflammatory cellular content. Healthy cells respond to death-inducing stimuli by imitating a variety of molecular pathways leading to cell death. Completion of the proper pathway is a critical cellular function to ensure that the appropriate outcome is ultimately achieved.
The quantification of the cell death response is an integral component of exploring cell biology, responses to cellular stress and performing high-throughput drug screens. One classic method of cell death detection is flow cytometry, which requires extensive handling of cells and only provides end-point data. Kinetic imaging, in contrast, is a critical application for studying dynamic biological processes in real time. Kinetic analysis of cell death analysis allows for sensitive, real-time determination of the accumulation of both apoptotic and necrotic events within the cellular population.
Here we present the use of apoptosis and necrosis dyes in combination with automated kinetic imaging to quantitatively assess the effects of known inducers of cell death in multiple cell lines. We use high contrast, label-free brightfield imaging to assay for total number of cells, and cellular dyes to label both apoptotic and necrotic cells concurrently. This allows for determination of percent apoptosis and necrosis in each population over forty-eight hours of drug treatment.