Category: Automation and High-Throughput Technologies
Apoptosis, or programmed cell death, is a highly regulated biological process which is initiated by the sequential activation of multiple cellular mechanisms. Apoptosis is ATP-dependent and its morphological changes are characterized by cell shrinkage, loss of plasma membrane integrity, chromatin condensation, nuclear fragmentation, and activation of a family of cysteine-containing, aspartate-directed proteases called caspases. It is a normal biological process that is necessary for evelopment of cells and cell cycle maturation. It is also important for maintaining the regular functions and activities of cells.
We have optimized a multi-parameter assay that measures four different apoptosis end points per well using the Multicyt Cell-Based kits. The four apoptosis end points that were measured in the assay are cell viability, caspase 3/7 activity, annexin V binding, and mitochondrial depolarization. Cell viability is determined by the ability of live cells to exclude DNA binding. The activation of Caspase 3/7 is detected by the use of NucView 488 Caspase-3/7 substrate, which upon cleavage by activated enzymes, results in an intracellular fluorescent signal. The membrane surface expression of externalized phosphatidylserine is detected by the binding of Annexin-V to the cellular surface and measured by the increase of fluorescent intensity. The mitochondrial membrane potential is determined by sequestration of a small fluorescent molecule inside the lumen of intact mitochondria with an active membrane potential. Upon mitochondrial depolarization, the dye leaks into the cytoplasm and is measured by a decrease in fluorescence. We screened two small molecule libraries and our preliminary data shows that the assay is robust in identifying agents that induce apoptosis.
Victoria Wong– Research Investigator, Pfizer, Groton, Connecticut
After I completed my post-doctoral training in GlaxoSmithKline Pharmaceuticals, I was a Sr. Principal Investigator in Lead Discovery Bristol-Myers Squibb, specialized in HTS. Currently I am working as a Sr. Principal Scientist in Hit Identification and Optimization at Pfizer and I am responsible for the global externalized HTS operation at Pfizer.