Category: Automation and High-Throughput Technologies
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 regimens1. However, the diversity of cell death modalities is complicated by shared signaling elements, overlapping mechanisms, and complex crosstalk among various cell death pathways2. 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.
Brad Larson– Principal Scientist, BioTek Instruments, Winooski, VT
Brad is a Principal Scientist at BioTek Instruments, INC., where he has worked since 2009. Prior to joining BioTek, he acquired extensive experience while employed in various capacities with multiple reagent providers. Brad’s current roles include optimizing new assay processes on BioTek’s line of automation, liquid handling, microplate detection, and imaging instrumentation. He has worked for more than 20 years with numerous automation and detection platforms, as well as a variety of cell models, to optimize 2D and 3D cell culture assays across multiple research fields. His current work has led to publications in Assay and Drug Development Technologies, The Journal of Laboratory Automation, The Journal of Biomolecular Screening, and Combinatorial Chemistry and High Throughput Screening, among others. Brad has additionally presented his work at numerous conferences across the United States, Europe, and Asia.