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Updated: Jul 15, 2026
Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells
Published on: August 26, 2021
High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins
Mamatha Sauermann1, Florian Hahne, Christian Schmidt
1Division of Molecular Genome Analysis, German Cancer Research Centre, Heidelberg, Germany.
Abstract:
After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.
Insights
Researchers developed a high-throughput flow cytometry assay to screen for proteins that trigger apoptosis (programmed cell death). This method successfully identified 6 novel apoptosis-inducing proteins from a pool of 200 candidates.
Area of Science:
- Proteomics
- Cell Biology
- Biotechnology
Background:
- The completion of the human genome sequence necessitates functional characterization of encoded proteins.
- Understanding protein function is crucial for identifying disease-related mechanisms.
Purpose of the Study:
- To develop and apply a high-throughput screening assay for identifying apoptosis-activating proteins.
- To systematically analyze protein functions and their relation to diseases.
Main Methods:
- A flow cytometry-based assay was developed to detect activated caspase-3 in cells overexpressing yellow fluorescent protein-tagged proteins.
- High-throughput screening was performed using a flow cytometer with an autosampler.
- Candidate proteins were validated using a microscopy-based nuclear fragmentation assay.
Main Results:
- A pilot screen of 200 proteins was conducted.
- The assay successfully identified proteins that induce apoptosis.
- Six novel apoptosis inducers were identified and validated.
Conclusions:
- The developed flow cytometry assay is effective for high-throughput screening of apoptosis-inducing proteins.
- This method facilitates the systematic functional analysis of proteins encoded by the human genome.
- The identified apoptosis inducers warrant further investigation for their roles in disease.
