Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*

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Unit Size: Cat No: Price (USD): Qty:
100 tests 13504 $345


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)534/619
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Peptidases and Proteases
Related Microplate Readers
Caspases play important roles in apoptosis and cell signaling. The activation of caspase-3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3 is also identified as a drug-screening target. Caspase 3 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This Amplite™ Caspase-3 Assay Kit uses Z-DEVD-ProRed™ as the fluorogenic indicator for assaying caspase-3 activity. Cleavage of R110 peptides by caspases generates strongly red fluorescent ProRed™ that can be monitored fluorimetrically at ~620 nm with excitation of ~530 nm. Z-DEVD-ProRed™ is recognized as the most sensitive red fluorogenic caspase 3/7 substrate. This kit can be used to continuously measure the activities of caspase-3 in cell extracts and purified enzyme preparations using a fluorescence microplate reader or fluorometer. It can also be used with flow cytometry for analyzing cell apoptosis and the activities of caspases 3 and 7.




Spectrum Advanced Spectrum Viewer

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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells with test compounds
  2. Add equal volume of caspase 3/7 working solution
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 535/620 nm

Important notes
Thaw Component A, B, C (if desired, Component D) at room temperature before use.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:535 nm
Emission:620 nm
Cutoff:610 nm
Recommended plate:Solid black
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.

1. Z-DEVD-ProRed™ stock solution (200X):
Add 65 µL of DMSO (not provided) into the vial of Component A.

2. (Optional) Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO directly to the vial of Ac-DEVD-CHO (Component D). This inhibitor can be used to confirm the correlation between fluorescence signal intensity and caspase 3/7-like protease activities.

Preparation of working solution

Add 50 μL of 200X Z-DEVD-ProRed™ stock solution and 100 μL of 1M DTT solution (Component C) into 10 mL Assay Buffer (Component B) and mix well.

 

Note: 50 μL of the 200X Z-DEVD-ProRed™ stock solution is enough for 100 assays using a reaction volume of 100 μL per assay.

Preparation of cell samples

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. Treat cells by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) into PBS or desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.

  2. Incubate the cell plates in an incubator for a desired period of time (3 - 5 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of caspase 3/7 working solution.

  4. Incubate the plate at room temperature for at least 1 hour, kept from light. Note: If desired, add 1 µL of the 1 mM stock solution of the caspase 3/7 Inhibitor Ac-DEVD-CHO into selected samples 10 minutes before adding the caspase 3/7 assay working solution at room temperature to confirm the caspase 3/7-like activities.

  5. Monitor the fluorescence intensity at Ex/Em = 535/620 nm (cut off at 610 nm) with either top or bottom read mode. Note: Sometimes, bottom read gives better signal to background ratio, centrifuge cell plate (especially for the nonadherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

High STMN1 level is associated with chemo-resistance and poor prognosis in gastric cancer patients
Authors: Tuya Bai, Takehiko Yokobori, Bolag Altan, Munenori Ide, Erito Mochiki, Mitsuhiro Yanai, Akiharu Kimura, Norimichi Kogure, Toru Yanoma, Masaki Suzuki
Journal: British Journal of Cancer (2017)

The trivalent cerium-induced cell death and alteration of ion flux in sweetpotato [Ipomoea batatas (L.) Lam]
Authors: Jiaojiao Jiang, Jianzhong Hu, Zeyi Xie, Qinghe Cao, Daifu Ma, Yonghua Han, Zongyun Li
Journal: Journal of Rare Earths (2017)

Uncarboxylated Osteocalcin Induces Antitumor Immunity against Mouse Melanoma Cell Growth
Authors: Yoshikazu Hayashi, Tomoyo Kawakubo-Yasukochi, Akiko Mizokami, Mai Hazekawa, Tomiko Yakura, Munekazu Naito, Hiroshi Takeuchi, Seiji Nakamura, Masato Hirata
Journal: Journal of Cancer (2017): 2478--2486

Docetaxel induces Bcl-2-and pro-apoptotic caspase-independent death of human prostate cancer DU145 cells
Authors: Takeharu Ogura, Yoshiyuki Tanaka, Hiroki Tamaki, Mamoru Harada
Journal: International journal of oncology (2016): 2330--2338

NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo
Authors: Lu Zhang, Jianjun Han, Amanda L Jackson, Leslie N Clark, Joshua Kilgore, Hui Guo, Nick Livingston, Kenneth Batchelor, Yajie Yin, Timothy P Gilliam
Journal: Journal of Hematology & Oncology (2016): 91

Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70
Authors: Matthew K Howe, Khaldon Bodoor, David A Carlson, Philip F Hughes, Yazan Alwarawrah, David R Loiselle, Alex M Jaeger, David B Darr, Jamie L Jordan, Lucas M Hunter
Journal: Chemistry & biology (2014): 1648--1659






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits
2. Cell Apoptosis & Proliferation

Certificate of Analysis