High-throughput live cell imaging of apoptosis
J C Puigvert1, Hans de Bont, Bob van de Water
1Leiden University, Leiden, The Netherlands.
Current Protocols in Cell Biology
|June 4, 2010
Summary
This study introduces a real-time method using automated microscopy and annexin V staining to track apoptosis kinetics in high-throughput screens. The protocol aids in identifying regulators of programmed cell death in response to toxic compounds.
Area of Science:
- Cell Biology
- Genomics
- Toxicology
Background:
- Apoptosis, or programmed cell death, is crucial for development and tissue health.
- Apoptosis signaling pathways are often investigated using functional genomics screens.
- Detecting apoptosis in real-time is essential for high-throughput toxicological and genetic screens.
Purpose of the Study:
- To develop and validate a kinetic method for real-time apoptosis detection.
- To facilitate high-throughput screening for apoptosis regulators and toxicant responses.
Main Methods:
- Utilized automated microscopy in a 96-well plate format.
- Employed fluorescent annexin V to label apoptotic cells via membrane changes.
- Integrated quantitative bioinformatics analysis for kinetic data.
Main Results:
- Successfully monitored the accumulation of apoptotic cells in real-time.
- Demonstrated the method's efficacy across multiple cell types and toxic compounds.
- Validated RNAi-based gene silencing effects on apoptosis kinetics.
Conclusions:
- The described protocol provides a robust, high-throughput method for kinetic apoptosis analysis.
- This approach is valuable for functional genomics and chemical screens investigating cell death pathways.
