Temporal dynamics of receptor-induced apoptosis in an affinity microdevice.
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 (D(2)R), 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 D(2)R 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.
Reif
Randall D
RD
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA.
Aguas
Charmaine
C
Martinez
Michelle M
MM
Pappas
Dimitri
D
D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002477:Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D011485:Protein Binding; D019014:fas Receptor / Q000378:metabolism*
D053222:Fas Ligand Protein; D019014:fas Receptor; D020169:Caspases