Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*
Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring Caspase 3 activation. Caspase 3 is widely accepted as a reliable indicator for cell apoptosis since the activation of caspase-3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This kit uses Z-DEVD-Rh 110-DVED-Z as a fluorogenic indicator for caspase-3 activity. Cleavage of Rh 110 peptides by caspase 3 generates strongly fluorescent Rh 110 that is monitored fluorimetrically at 520-530 nm with excitation of 480-500 nm. The kit provides all the essential components with an optimized assay protocol. The assay is robust, and can be readily adapted for high-throughput assays. Using 100 uL of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 200 assays. Using 25 uL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 800 assays.
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 3/7 Substrate working solution
- Incubate at room temperature for 1 hour
- Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 50 µL of Caspase 3/7 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 3/7 Substrate working solution. Note: Aliquot and store the unused Caspase 3/7 Substrate (Components A) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles
SAMPLE EXPERIMENTAL PROTOCOL
Cells preparation:
- For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well plate or 5,000cells/well/20mL for a 384-well plate.
- For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 cells/well/90 uL for a 96-well poly-D lysine plate or 20,000 cells/well/20 uL for a 384-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
Sample 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 37°C, 5% CO2, incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 Substrate working solution.
- Incubate the plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-DEVD-CHO caspase 3/7 inhibitor into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the inhibition of the caspase 3/7-like activities.
- Centrifuge 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 = 490/525 nm (Cutoff = 515 nm).
Spectrum
Open in Advanced Spectrum Viewer
Product family
Name | Excitation (nm) | Emission (nm) |
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence* | 341 | 441 |
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence* | 532 | 619 |
Citations
View all 36 citations: Citation Explorer
High-dose ascorbate exerts anti-tumor activities and improves inhibitory effect of carboplatin through the pro-oxidant function pathway in uterine serous carcinoma cell lines
Authors: Shen, Xiaochang and Wang, Jiandong and Deng, Boer and Chen, Shuning and John, Catherine and Zhao, Ziyi and Sinha, Nikita and Haag, Jennifer and Sun, Wenchuan and Kong, Weimin and others,
Journal: Gynecologic Oncology (2024): 93--102
Authors: Shen, Xiaochang and Wang, Jiandong and Deng, Boer and Chen, Shuning and John, Catherine and Zhao, Ziyi and Sinha, Nikita and Haag, Jennifer and Sun, Wenchuan and Kong, Weimin and others,
Journal: Gynecologic Oncology (2024): 93--102
Hexamethylene Amiloride synergizes with Venetoclax to induce lysosome-dependent cell death in acute myeloid leukemia
Authors: Jiang, Xinya and Huang, Kexiu and Sun, Xiaofan and Li, Yue and Hua, Lei and Liu, Fangshu and Huang, Rui and Du, Juan and Zeng, Hui
Journal: iScience (2023)
Authors: Jiang, Xinya and Huang, Kexiu and Sun, Xiaofan and Li, Yue and Hua, Lei and Liu, Fangshu and Huang, Rui and Du, Juan and Zeng, Hui
Journal: iScience (2023)
Apical-Out Chicken Enteroids as an In-Vitro Model to Study Effects of an Alternative Growth Promoter on Indicators of Gut Health
Authors: Tamazian, Lorik
Journal: (2023)
Authors: Tamazian, Lorik
Journal: (2023)
Metformin sensitizes AML cells to venetoclax through endoplasmic reticulum stress—CHOP pathway
Authors: Hua, Lei and Yang, Nianhui and Li, Yue and Huang, Kexiu and Jiang, Xinya and Liu, Fangshu and Yu, Zhi and Chen, Jie and Lai, Jing and Du, Juan and others,
Journal: British Journal of Haematology (2023)
Authors: Hua, Lei and Yang, Nianhui and Li, Yue and Huang, Kexiu and Jiang, Xinya and Liu, Fangshu and Yu, Zhi and Chen, Jie and Lai, Jing and Du, Juan and others,
Journal: British Journal of Haematology (2023)
GONIOTHALAMIN INHIBITS CELL GROWTH, PERTURBS CELL CYCLE AND INDUCES APOPTOSIS IN HUMAN OSTEOSARCOMA SAOS-2 CELLS: Received 2023-01-03; Accepted 2023-02-15; Published 2023-06-06
Authors: Bakar, Siti Aishah Abu and Azhar, Nur Asna and Mohamad, Noor Muzamil and Nordin, Ira Maya Sophia and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Ahmad, Nor Hazwani
Journal: Journal of Health and Translational Medicine (JUMMEC) (2023): 105--115
Authors: Bakar, Siti Aishah Abu and Azhar, Nur Asna and Mohamad, Noor Muzamil and Nordin, Ira Maya Sophia and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Ahmad, Nor Hazwani
Journal: Journal of Health and Translational Medicine (JUMMEC) (2023): 105--115
Page updated on October 4, 2024