Feb 10, 2025
Caspase-3/7 activity
- 1Stanford University
Protocol Citation: Ching-Chieh Chou, Judith Frydman 2025. Caspase-3/7 activity. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv52ow4v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 07, 2025
Last Modified: February 10, 2025
Protocol Integer ID: 119805
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Aligning Science Across Parkinson's
Grant ID: ASAP-024268
Abstract
Detecting cell apoptosis by measuring Caspase-3/7 activation
Protocol for detecting Caspase-3/7 activation
Protocol for detecting Caspase-3/7 activation
Caspase-3/7 substrate FAM-DEVD-FMK (ImmunoChemistry) is reconstituted in DMSO and stored at -20°C. The FAM-DEVD-FMK reagent is one of the fluorochrome-labeled inhibitors of caspases that covalently and irreversibly binds to the active caspases. The green fluorescence intensity is a direct measurement of Caspase-3/7 activity.
To test toxic or rescuing effects on cell apoptosis, the cells are pre-treated with DMSO or any pharmacological perturbations.
When cells are available, first dilute the 150X stock concentrate 1:5 in PBS to form the staining solution at 30X.
Add the 30X staining solution at a dilution of 1:30 to cell culture medium to form 1X staining solution. The solution should be used immediately.
Prepare 1X Apoptosis Buffer by diluting 10X Apoptosis Buffer 1:10 in dH2O.
Add 100-300 µL FAM-FLICA solution to each well and incubate for 30 min at 37°C.
Aspirate the FAM-FLICA staining solution and rinse 1X Apoptosis Buffer once.
The cells are fixed by 4% paraformaldehyde (PFA) for 15 min and stained with antibodies and 5 μg/mL Hoechst for confocal microscope imaging.