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Updated: May 13, 2026
Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
Published on: March 24, 2023
Bioluminescence determination of active caspase-3 in single apoptotic cells
Marcela Lišková1, Karel Klepárník, Eva Matalová
1Institute of Analytical Chemistry, v.v.i., Czech Academy of Science, Brno, Czech Republic.
Abstract:
Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.
Insights
Researchers developed a sensitive method to detect active caspase-3 in single cells. This bioluminescence technology allows for precise measurement of apoptosis, crucial for understanding cell death in development and disease.
Area of Science:
- Biochemistry
- Cell Biology
- Biotechnology
Background:
- Apoptosis is programmed cell death vital for development and homeostasis.
- Dysregulation of apoptosis is implicated in cancer and autoimmune diseases.
- Understanding apoptosis at the single-cell level is critical for biological and medical research.
Purpose of the Study:
- To develop a high-sensitivity instrumentation and protocol for detecting active caspase-3 in individual mammalian cells.
- To investigate apoptosis pathways at the single-cell level.
Main Methods:
- Utilized a specific cleavage of modified luciferin by caspase-3, initiating a bioluminescence reaction.
- Employed a photomultiplier tube in photon counting mode for sensitive photon detection.
- Compared three different instrumental setups for caspase-3 determination in cells and tissue samples.
Main Results:
- Developed a highly sensitive detection system for active caspase-3.
- Achieved detection of as little as 6.5 fg (122,000 molecules) of caspase-3 in individual apoptotic mouse cells using a miniaturized system.
- Demonstrated the capability to analyze caspase-3 activity in single cells.
Conclusions:
- The developed technology enables sensitive detection of active caspase-3 in individual apoptotic cells.
- This advancement facilitates detailed investigation of apoptosis at the single-cell level.
- The method holds potential for biological research and clinical diagnostics related to apoptosis-driven diseases.
