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

Additional fluorescent color(s): 
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Unit Size: Cat No: Price (USD): Qty:
100 tests 13504 $345

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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Ex/Em (nm)534/619
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.

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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

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.
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 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)

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

Certificate of Analysis