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ApoSight™ Green Caspase 3/7 substrate
ApoSight™ Green Caspase 3/7 substrate is the first fluorogenic probe for the direct fluorescence imaing of caspase activities in live cells. It consists of three moieties including a). a masked fluorophore, b). a caspase-selective peptide fragment (DEVD), and c). a cell-penetrating moiety. The cell-penetrating moiety carries the probe into live cells. Upon entering live cells, the caspase-selective peptide fragment is cleaved by a caspase to release the masked fluorophore. The intensity of recovered fluorescence is directly related to the activity of the caspase 3/7 to be measured. Compared to the existing caspase assays in live cells, ApoSight™ Green Caspase 3/7 substrate is much more robust, convenient, and accurate. ApoSight™ Green Caspase 3/7 substrate releases a fluorophore that has Ex/Em ~490/520 nm. It does not need a DNA interaction to be fluorescent as reported for NucView reagents. It does not inhibit caspase activity as reported for the FMK peptide probes. Although fluorescent FMK peptide inhibitors of caspases are widely used for detecting caspase activities in live cells, this technology has a few severe limitations: a). FMK caspase inhibitors have high cytotoxicity since FMK peptides bind covalently to active caspases; b). The irreversible covalent binding of FMK peptides to caspases inhibits caspase activities, causing false positive apoptosis; c). FMK assays have extremely high background, and require intensive washings, resulting in extremely low throughput; d). FMK peptides are not stable in aqueous solutions and must be used immediately. ApoSight™ Green Caspase 3/7 substrate can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds at a density of 5 × 104 to 2 × 105 cells/100 µL/well for 96-well plate
- Add equal volume of ApoSight™ Caspase 3/7 Substrate working solution
- Incubate in a 5% CO2 incubator at 37 °C for 60 minutes
- Wash the cells 1-2 time with HHBS
- Analyze the cells with flow cytometer with 530/30 nm filter (FITC channel) or with fluorescence microscope with FITC filter set
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note Aliquot in single use aliquots to avoid repeated freeze-thaw cycle. Protect from light.
ApoSight™ Green Caspase 3/7 Substrate stock solution (200X)
Add 50 µL of DMSO into the vial of ApoSight™ Green Caspase 3/7 Substrate to make 200X ApoSight™ Green Caspase 3/7 Substrate stock solution.Note Aliquot in single use aliquots to avoid repeated freeze-thaw cycle. Protect from light.
PREPARATION OF WORKING SOLUTION
ApoSight™ Green Caspase 3/7 Substrate working solution
Prepare ApoSight™ Green Caspase 3/7 Substrate working solution by mixing 5 µL of 200X ApoSight™ Green Caspase 3/7 Substrate stock solution with 1 mL of Hanks and 20 mM Hepes buffer (HHBS, Cat# 20011) or buffer of your choice and mix well.Note 100 µL of ApoSight™ Green Caspase 3/7 Substrate working solution is enough for 10 tests in a 96-well plate format.
Note Prepare enough ApoSight™ Green Caspase 3/7 Substrate working solution right before the experiment, and use promptly.
SAMPLE EXPERIMENTAL PROTOCOL
Examples for inducing apoptosis in suspension culture
Treat Jurkat cells with 2 μg/mL camptothecin for 3 hoursTreat Jurkat cells with 1 μM staurosporine for 3-4 hours
Treat HL-60 cells with 4 μg/mL camptothecin for 4 hours
Treat HL-60 cells with 1 μM staurosporine for 4 hours.
Note Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
Sample protocol for Fluorescence Microscopy
- Prepare cells with test compounds at a density of 5 × 104 to 2 × 105 cells/100 µL/well/96-well plate.
- Add equal volume of Caspase 3/7 Substrate working solution to the cells (100 µL/well/96 well-plate).
- Incubate in a 5% CO2 incubator at 37 °C for 60 minutes.
- Wash cells 1-2 times with HHBS or buffer of your choice.
- Image with a fluorescence microscope using a FITC filter set.
Sample protocol for Flow Cytometry
- Add 200 X ApoSight™ Green Caspase 3/7 Substrate stock solution into the cell solution at a 1:200 ratio, and incubate the cells in a 5% CO2 incubator at 37 °C for 60 minutes.
Note The cells can be concentrated up to ~5 X 106 cells/mL for ApoSight™ Green Caspase 3/7 Substrate labeling. The appropriate incubation time depends on the individual cell type and cell concentration used. - Monitor the fluorescence intensity with a flow cytometer using 530/30 nm filter (FITC channel).
Note To increase the signal/background ratio, spin down the cells at ~200 g for 5 minutes, wash cells with 1 mL washing buffer such as HHBS or buffer of your choice ones, and resuspend the cells in the desired amount of washing.
Spectrum
References
View all 4 references: Citation Explorer
Cell culture conditions affect the ability of high content imaging assay to detect drug-induced changes in cellular parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
Authors: Balasubramanian, Bharathi and Belak, Vaclav and Verma, Isha and Prysiazhniuk, Yeva and Sannajust, Frederick and Trepakova, Elena S
Journal: Toxicology reports (2019): 305-320
Authors: Balasubramanian, Bharathi and Belak, Vaclav and Verma, Isha and Prysiazhniuk, Yeva and Sannajust, Frederick and Trepakova, Elena S
Journal: Toxicology reports (2019): 305-320
In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy.
Authors: Guisier, Florian and Bohn, Pierre and Patout, Maxime and Piton, Nicolas and Farah, Insaf and Vera, Pierre and Thiberville, Luc and Salaün, Mathieu
Journal: PloS one (2017): e0180576
Authors: Guisier, Florian and Bohn, Pierre and Patout, Maxime and Piton, Nicolas and Farah, Insaf and Vera, Pierre and Thiberville, Luc and Salaün, Mathieu
Journal: PloS one (2017): e0180576
Polyphenols act synergistically with doxorubicin and etoposide in leukaemia cell lines.
Authors: Mahbub, A A and Le Maitre, C L and Haywood-Small, S L and Cross, N A and Jordan-Mahy, N
Journal: Cell death discovery (2015): 15043
Authors: Mahbub, A A and Le Maitre, C L and Haywood-Small, S L and Cross, N A and Jordan-Mahy, N
Journal: Cell death discovery (2015): 15043
Monitoring cleaved caspase-3 activity and apoptosis of immortalized oligodendroglial cells using live-cell imaging and cleaveable fluorogenic-dye substrates following potassium-induced membrane depolarization.
Authors: Smith, Graham S T and Voyer-Grant, Janine A M and Harauz, George
Journal: Journal of visualized experiments : JoVE (2012)
Authors: Smith, Graham S T and Voyer-Grant, Janine A M and Harauz, George
Journal: Journal of visualized experiments : JoVE (2012)
Page updated on November 19, 2024
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