Apoptosis, a process in which a cell systematically triggers its own death, is widely utilized in the body. Malfunction of the apoptosis process can lead to serious health problems such as cancer. There are two major pathways for the cell to undergo apoptosis, extrinsic and intrinsic, dependent on the method of induction. The goal of this research is to elucidate and compare the temporal dynamics of apoptosis in Jurkat T-Lymphocytes with respect to caspase activity, through both the intrinsic and extrinsic pathways using fluorescence microscopy. Microfluidic devices were fabricated with a channel coated with antibodies to trap cells in a controlled environment over the course of the studied period (6 hours). The extrinsic pathway studies used anti-CD95 to capture the cells and induce apoptosis. The experiments examining the intrinsic pathway used anti-CD71 to capture the cells while they were exposed to 0.05 mM hydrogen peroxide to induce apoptosis. The fluorogenic caspase probe, L-bisaspartic acid Rhodamine 110 (D2R) was used to visualize general caspase activity while derivatives of the probe (DEVD2R and IETD2R) were used to monitor caspases-3 and caspase-8. For the intrinsic pathway, overall caspase activity initiated from 4.3 to 5.3 hours after induction, lasting 40 to 60 minutes, while caspase-3 activity started 5.3 hours after induction and lasted 20 to 40 minutes. For the extrinsic pathway, overall caspase activity started 3.3 to 5.3 hours after induction and lasted between 1.3 – 4.6 hours, caspase-8 activity was shown to start 1.6 hours after induction and last for 180 minutes, and caspase-3 activity was shown to start 5.3 hours after induction and lasted 20 to 40 minutes. The similarities and differences between the timing of caspase activation in the two different pathways will be discussed as this knowledge could be helpful when designing therapies that affect apoptosis.

Exploring the temporal dynamics of caspase activity during different pathways of apoptosis

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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