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Updated: Apr 27, 2026
Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
Published on: March 24, 2023
A miniaturized device for bioluminescence analysis of caspase-3/7 activity in a single apoptotic cell
Eva Adamová1, Marcela Lišková, Eva Matalová
1Institute of Analytical Chemistry, v.v.i., Czech Academy of Sciences, Veveří 97, 602 00, Brno, Czech Republic.
Abstract:
Caspases are key enzymes activated during the apoptotic machinery. Apoptosis as a way of programmed cell death becomes deregulated in some pathologies including cancer transformations, neurodegenerative, or autoimmune diseases. Most of the methods available for the detection of apoptosis and caspases provide qualitative information only or quantification data as an average from cell populations or cell lysates. Several reports point to the importance of more accurate single-cell analyses in biomedical studies due to heterogeneity at tissue as well as cell level. To meet these requirements, we developed a miniaturized device enabling detection and quantification of active caspase-3/7 in individual cells at a femtogram level (10(-15) g). The active caspase-3/7 detection protocol is based on the bioluminescence chemistry commercially available as a Caspase-Glo™ 3/7 reagent developed by Promega. As a model, we used human stem cells treated by camptothecin to induce apoptosis. Individual apoptotic cells were captured from a culture medium under a microscope and transferred by a micromanipulation system into a detection capillary containing 2 μl of the reagent. Cells without activation by camptothecin served as negative controls. The detection limit of active caspase-3/7 achieved in the miniaturized system was determined as 0.20 and limit of quantification as 0.65 of the amount found in a single apoptotic human stem cell. Such a sensitive method could have a wide application potential in laboratory medicine and related clinically oriented research.
Insights
This study introduces a novel miniaturized device for precise, single-cell quantification of active caspase-3/7, crucial enzymes in apoptosis. This advancement enables femtogram-level detection, offering new possibilities for disease research.
Area of Science:
- Biochemistry
- Cell Biology
- Biotechnology
Background:
- Caspases are critical enzymes in apoptosis, a programmed cell death pathway.
- Dysregulation of apoptosis is implicated in diseases like cancer, neurodegeneration, and autoimmune disorders.
- Current methods for caspase detection offer limited single-cell resolution, hindering accurate analysis of cellular heterogeneity.
Purpose of the Study:
- To develop a miniaturized system for sensitive, single-cell detection and quantification of active caspase-3/7.
- To address the need for high-resolution analysis of apoptotic processes at the individual cell level.
Main Methods:
- A miniaturized device was engineered for active caspase-3/7 detection.
- The system utilizes bioluminescence chemistry with a commercially available Caspase-Glo™ 3/7 reagent.
- Human stem cells induced into apoptosis were analyzed using micromanipulation and the developed device.
Main Results:
- The system achieved femtogram-level (10^-15 g) detection and quantification of active caspase-3/7 in individual cells.
- The detection limit was determined to be 0.20 and the limit of quantification as 0.65 of the amount in a single apoptotic human stem cell.
- The method demonstrated high sensitivity for analyzing apoptosis at the single-cell level.
Conclusions:
- The developed miniaturized device enables highly sensitive, quantitative analysis of active caspase-3/7 in single cells.
- This technology has significant potential for applications in laboratory medicine and clinical research, particularly for studying diseases with deregulated apoptosis.
- The ability to analyze apoptotic markers at the single-cell level overcomes limitations of population-based assays and accounts for cellular heterogeneity.
