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Updated: Jun 15, 2026
Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
Published on: March 5, 2018
Time-lapse, single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during
Abstract:
Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.
Insights
This study introduces a cell-based bioimaging technique to monitor apoptosis, a key process for anti-cancer drug development. The method visualizes caspase-3 activation and membrane changes in real-time, offering a novel approach beyond traditional biochemical assays.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Apoptosis is crucial for evaluating anti-cancer drug efficacy.
- Conventional assays for apoptosis primarily focus on biochemical events.
- Existing methods lack real-time cellular visualization of apoptotic processes.
Purpose of the Study:
- To investigate staurosporine (STS)-induced apoptosis in murine macrophage RAW-264.7 cells using cell-based bioimaging.
- To visualize and monitor key apoptotic events, including caspase-3 activation and membrane integrity changes.
- To compare bioimaging findings with conventional biochemical assays.
Main Methods:
- Utilized time-lapse confocal microscopy for real-time monitoring of apoptosis.
- Tracked caspase-3 activation via green fluorescent protein translocation to the nucleus.
- Visualized phosphatidylserine (PS) externalization using annexin-V and fluorescein isothiocyanate.
- Assessed plasma membrane disintegration with propidium iodide nuclear dye.
Main Results:
- Caspase-3 activation and membrane blebbing were observed 5 hours after 1 μM STS treatment.
- Early apoptosis was detected by monitoring externalized PS using annexin-V.
- Late apoptosis was confirmed by plasma membrane disintegration visualized with propidium iodide.
- Bioimaging data correlated well with biochemical assays for caspase activation and membrane integrity.
Conclusions:
- Cell-based bioimaging provides a powerful, real-time method to study apoptosis.
- This technique offers a comprehensive view of apoptotic events, complementing traditional assays.
- The findings support the utility of bioimaging in anti-cancer drug candidate evaluation.
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