High-throughput live cell imaging of apoptosis
J C Puigvert1, Hans de Bont, Bob van de Water
1Leiden University, Leiden, The Netherlands.
Abstract:
Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant-induced cell injury commonly use apoptosis as an end-point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high-throughput screens is described. The method uses automated microscopy in a 96-well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase-dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi-based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens.
Insights
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.
