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.

Primary Author - February Poster(s)

• VW

  Victoria Wong

  – Research Investigator, Pfizer, Groton, Connecticut