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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 to maintain a homeostatic quantity of cells. Malfunctions in this process can result in numerous consequences, particularly tumor development. For a cell to undergo apoptosis, there are two major pathways, dependent upon the method of induction: the extrinsic and intrinsic pathways. The goal of this research is to elucidate the temporal dynamics of apoptosis in Jurkat T-lymphocytes with respect to caspase activity in the extrinsically-activated pathway, using fluorescence microscopy. Microfluidic devices were fabricated from polydimethylsiloxane (PDMS) and contained an antibody-coated channel. The devices were used for cellular capture of the lymphocytes to trap and observe cells in a controlled, sustainable- environment for a given duration. The antibody coated in the channel, anti-CD95, induces apoptosis through Fas receptor binding. The result of the antibody binding is the activation of a caspase enzyme, creating a destructive caspase cascade. General caspase activation was visualized over a 9-hour period with the fluorogenic caspase probe L-bisaspartic acid Rhodamine 110 (D2R). Derivatives of the fluorogenic probe containing sequences to specific caspases were used to monitor the first and last caspases to be activated in the apoptotic pathway. The derivative (IETD)2R was used to monitor the primary initiator caspase activation (caspase-8) and the (DEVD)2R for executioner caspase activation (caspase-3). The overall average caspase activity began 2.4 ± 0.6 hours post-induction and lasted 360 ± 60 minutes, caspase-8 activity was shown to start 1.8 ± 0.8 hours after induction and remain active for 120 ± 40 minutes, and caspase-3 activity started 5.2 ± 1.0 hours after induction, lasting 130 ± 60 minutes. Timing of caspase activation is an important field of study as it could be helpful in designing apoptosis-targeted therapies.
Zwemer, Catherine, "An Extended View of the Temporal Dynamics of Caspase Activity During the Extrinsic Pathway of Apoptosis in Jurkat T-Lymphocytes" (2020). Student Research Submissions. 343.