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An Automated Approach to Kinetic, Live Cell Multi-Parametric Cell Death AnalysesDownload
February 05, 2018
Author: Brad Larson, BioTek Instruments, Inc., Winooski, VT USA
Deep insight into regulation and dysregulation of cell death processes is critical towards understanding disease states such as cancer, and developing effective, well-tolerated treatment therapies. In fact, many programmed and non-programmed cell death pathways are being studied for the development of more effective and less toxic chemotherapeutic regimens. However, the diversity of cell death modalities is complicated by shared signaling elements, overlapping mechanisms, and complex crosstalk among various cell death pathways. Differentiating the morphological hallmarks of cell death pathways can be labor intensive, and when incorporating end-point assays, can often miss critical yet transient events.
Here, we demonstrate an automated, multiplexed method to assess real-time cell death. Three common cell death biomarkers are measured: mitochondria membrane potential, phosphatidylserine (PS) externalization, and cell membrane integrity, using fluorescent probes from Abcam. The fluorescent, positively-charged tetramethylrhodamine ethyl ester (TMRE) dye readily passes through cell membranes and accumulates in healthy, active mitochondria, where it produces a red-orange signal. If the mitochondria membrane is depolarized or inactive, as in apoptotic and necrotic cells, the dye diffuses throughout the cell. The green fluorescent probe, pSIVA™-IANBD binds to the non-polar environment of the cell’s membrane lipid bilayer, and detects irreversible and transient phosphatidylserine exposure that is characteristic of apoptosis and necroptosis. Finally, the far-red fluorescent dye, DRAQ7™ is impermeant in healthy cells, while it stains nuclei in dead and permeabilized necrotic and necroptotic cells. Combining these dyes into a single, multiplexed method with real-time morphological analysis provides major advantages when characterizing cell death systems. Fibrosarcoma target cells and dyes were combined in a microplate along with a known inhibitor compound, and incubated in an automated benchtop incubator. The plates were automatically transferred from the incubator to a combined microplate reader and automated digital imager every two hours for a total of forty-eight hours, where fluorescent imaging was performed to assess cellular activity, as well as high-contrast brightfield imaging to allow for accurate cell counting over the entire incubation period.