Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*
Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used. This particular kit is designed to monitor cell apoptosis by measuring caspase 9 activity. Caspase 9 is a member of the CED-3 subfamily. Activated Caspase-9 cleaves downstream caspases such as caspase-3, -6 and -7, initiating the caspase cascade. It is essential for apoptosis during normal development of the central nervous system. Caspase 9 is proven to have selectivity for the peptide sequence Leu-Glu-His-Asp (LEHD). This kit uses Ac-LEHD-AMC as a fluorogenic indicator for caspase 9 activity. Cleavage of AMC by caspase 9 generates strongly fluorescent AMC. The kit provides all the essential components. The assay is robust and can be readily adapted for high throughput screening. It can be used to either quantify the activated caspase 9 activities in apoptotic cells or screen the caspase 9 inhibitors. Quite a few labs have used this kit for high throughput screenings.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Add equal volume of Caspase 9 Substarte working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Incubate at room temperature for 30 - 60 minutes
- Monitor the fluorescence at Ex/Em = 375/435 nm (Cutoff = 420 nm)
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 50 μL of Caspase 9 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 9 Substrate working solution. Protect from light. Note: Aliquot and store the unused Caspase 9 Substrate (Component A) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells by adding 10 µL/well of 10X test compounds (96-well plate) or 5 µL/well of 5X test compounds (384-well plate) into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
- Incubate the cell plate in a 5% CO2, 37°C incubator for a desired period of time (4-6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 9 Substrate working solution.
- Incubate the Caspase 9 Substrate working solution plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-LEHD-CHO caspase 9 inhibitor to selected samples 10 minutes before adding Caspase 9 Substrate working solution at room temperature to confirm the inhibition of the caspase 9-like activity.
- Centrifuge the cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
- Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 375/435 nm (Cutoff = 420 nm).
Spectrum
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence* | 500 | 522 | 80000 |
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence* | 532 | 619 | - |
Citations
View all 18 citations: Citation Explorer
Involvement of necroptosis in the selective toxicity of the natural compound ($\pm$) gossypol on squamous skin cancer cells in vitro
Authors: Haasler, Lisa and von Montfort, Claudia and Kondadi, Arun Kumar and Golombek, Mathias and Ebbert, Lara and Wenzel, Chantal-Kristin and Stahl, Wilhelm and Reichert, Andreas S and Brenneisen, Peter
Journal: Archives of Toxicology (2023): 1--18
Authors: Haasler, Lisa and von Montfort, Claudia and Kondadi, Arun Kumar and Golombek, Mathias and Ebbert, Lara and Wenzel, Chantal-Kristin and Stahl, Wilhelm and Reichert, Andreas S and Brenneisen, Peter
Journal: Archives of Toxicology (2023): 1--18
Contact-dependent signaling triggers tumor-like proliferation of CCM3 knockout endothelial cells in co-culture with wild-type cells
Authors: Rath, Matthias and Schwefel, Konrad and Malinverno, Matteo and Skowronek, Dariush and Leopoldi, Alexandra and Pilz, Robin A and Biedenweg, Doreen and Bekeschus, Sander and Penninger, Josef M and Dejana, Elisabetta and others,
Journal: Cellular and Molecular Life Sciences (2022): 1--20
Authors: Rath, Matthias and Schwefel, Konrad and Malinverno, Matteo and Skowronek, Dariush and Leopoldi, Alexandra and Pilz, Robin A and Biedenweg, Doreen and Bekeschus, Sander and Penninger, Josef M and Dejana, Elisabetta and others,
Journal: Cellular and Molecular Life Sciences (2022): 1--20
Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7
Authors: Bakar, Siti Aishah Abu and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Azhar, Nur Asna and Mohamad, Noor Muzamil and Ahmad, Nor Hazwani
Journal: BioMed Research International (2022)
Authors: Bakar, Siti Aishah Abu and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Azhar, Nur Asna and Mohamad, Noor Muzamil and Ahmad, Nor Hazwani
Journal: BioMed Research International (2022)
Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells
Authors: Chang, Ying-Tzu and Teng, Yu-Ning and Lin, Kun-I and Wang, Charles CN and Morris-Natschke, Susan L and Lee, Kuo-Hsiung and Hung, Chin-Chuan
Journal: Scientific reports (2019): 1--11
Authors: Chang, Ying-Tzu and Teng, Yu-Ning and Lin, Kun-I and Wang, Charles CN and Morris-Natschke, Susan L and Lee, Kuo-Hsiung and Hung, Chin-Chuan
Journal: Scientific reports (2019): 1--11
Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
Page updated on October 30, 2024