Temporal dynamics of receptor-induced apoptosis in an affinity microdevice |
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Authors: | Randall D Reif Charmaine Aguas Michelle M Martinez Dimitri Pappas |
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Institution: | (1) Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA; |
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Abstract: | The temporal dynamics of Fas-induced apoptosis is elucidated. Jurkat cells are captured on the affinity surface of a microdevice
coated with anti-CD95, an antibody known to induce apoptosis in cells via the extrinsic (caspase 8) pathway. The timing of
apoptosis induction is controlled by the binding of the cells to the surface. Once bound, the cells are continuously stained
with the caspase probe, l-bisaspartic acid rhodamine 110 (D2R), and the fluorescence of the cells was monitored for 6 h by light microscopy. This approach normalizes the temporal dynamics
for each cell, as the binding event is also the start of apoptosis. In addition to providing the number of apoptotic cells
over time, the fluorescence of individual cells can be monitored, providing information about the timing of caspase activity
in each cell. The rate of caspase cleavage of D2R in each cell is also measured and shows good agreement between the cells in a given population. The effects of the caspase
inhibitor, z-VAD-FMK, on the timing of caspase activity are also investigated and are shown to dramatically slow the apoptotic
process. In the future, other caspase probes could be used to provide additional information about the temporal dynamics of
caspase activation. Additional techniques, such as fluorescence correlation spectroscopy, can be coupled to these methods
to provide faster temporal response and help to elucidate the heterogeneity of the apoptosis process. |
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