Temporal Dynamics of Apoptotic Activity in Jurkat Cells

Abstract

Apoptosis, a process in which a cell systematically triggers its own death in response to DNA damage or external stimuli, is widely utilized in the body. Malfunction of the apoptosis process can lead to serious health problems such as cancer. There are several known pathways that execute apoptosis utilizing a family of enzymes called Caspases. The (intrinsic) pathway is the focus of this research, and initiates caspase activity through mitochondrial depolarization.The goal of this research project was to find suitable initiators of apoptosis in Jurkat T‐Lymphocytes and elucidate their temporal dynamics with respect to caspase activity. Microfluidic devices were fabricated to capture cells and view this process. To identify suitable inducers of apoptosis to use, cells were exposed to several compounds and monitored over six hour time periods using Fluorescence Microscopy. Caspase activation was confirmed with the use of a caspase‐specific fluorogenic probe, L‐bisaspartic acid rhodamine 110.Anti‐CD95, staurosporine, and hydrogen peroxide were used as inducers of apoptosis resulting in induction rates of 8.88%, 1.66%, and 1.24% with un‐induced rates of 0.59%, 1.88%, and 0.24% respectively. On a microfluidic device, cells were induced with Hydrogen Peroxide and their fluorescence intensity was measured over time. Apoptotic cells with active caspase enzymes consistently reached peak intensity two hours after caspase activation began. Using these results, mitochondrial depolarization will be tracked simultaneously with caspase activity utilizing MitoTracker Red.Support or Funding InformationThis research would not be possible without support from the Department of Chemistry at the University of Mary Washington, as well as a grant from the University of Mary Washington Summer Science Institute.