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Amplite® Colorimetric Caspase 3/7 Assay Kit *Yellow Color*

Detection of caspase 3/7 Activity in Jurkat cells. The cells were treated with staurosporine (SS) at the concentration of 0-1 µM for 4 hours at 37ºC. After treatment, cells were incubated with caspase 3/7 assay solution for 2 hours. The absorbance was measured at 490 nm using a SpectraMax reader (Molecular Devices).
Detection of caspase 3/7 Activity in Jurkat cells. The cells were treated with staurosporine (SS) at the concentration of 0-1 µM for 4 hours at 37ºC. After treatment, cells were incubated with caspase 3/7 assay solution for 2 hours. The absorbance was measured at 490 nm using a SpectraMax reader (Molecular Devices).
Detection of caspase 3/7 Activity in Jurkat cells. The cells were treated with staurosporine (SS) at the concentration of 0-1 µM for 4 hours at 37ºC. After treatment, cells were incubated with caspase 3/7 assay solution for 2 hours. The absorbance was measured at 490 nm using a SpectraMax reader (Molecular Devices).
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Spectral properties
Extinction coefficient (cm -1 M -1)80000
Excitation (nm)500
Emission (nm)522
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22


Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Caspases play important roles in apoptosis and cell signaling. The activation of Caspase 3/7 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3/7 is also identified as a drug-screening target. Caspase inhibitors have anti-cancer and other pharmalogical potentials. It has been proven that Caspase 3/7 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). Our Amplite® Colorimetric Caspase 3/7 Assay Kit uses (Z-DEVD)2R110 as the chromogenic indicator for assaying caspase 3/7 activity. R110 peptide sustrates are colorless. Cleavage of R110 peptides by caspases generates R110, a yellow color dye that can be monitored at 490-520 nm. The increase in the absorbance of caspase-induced R110 is proportional to the activities of caspases. This kit can be used to continuously measure the activities of caspase 3/7 in cell extracts and purified enzyme preparations with an absorbance microplate reader with much higher sensitivity than the other commercial kits that use DEVD-pNA peptide.


Absorbance microplate reader

Absorbance490 nm
Recommended plateClear bottom


Example protocol


Protocol Summary
  1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  2. Add equal volume of Caspase 3/7 working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at room temperature for 1 - 2 hours
  4. Monitor absorbance at 490 nm 


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


Add 50 μL of 200X Caspase 3/7 Substrate stock solution (Component A) into 10 mL Assay Buffer (Component B), and mix well to make Caspase 3/7 working solution. Note: This Caspase 3/7 working solution is enough for 100 assays using a reaction volume of 100 μL per assay. Before opening the vial, do brief centrifuge to accumulate the stock solution to the bottom of the tube.


  1. Treat cells with 10 µL of 10X test compounds (for a 96-well plate) or 5 µL of 5X test compound (for a 384-well plate) in PBS or desired buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
  2. Incubate the cell plates in an incubator for a desired period of time to induce apoptosis. Note: We treated Jurkat cells with staurosporine (SS) for 4 hours at 37°C to induce cell apoptosis. See Figure 1 for details.
  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, protected from light.
  5. Centrifuge cell plates at 800 rpm for 2 minutes with brake off.
  6. Monitor the absorbance increase with an absorbance plate reader at OD =490 nm. 


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Spectral properties

Extinction coefficient (cm -1 M -1)80000
Excitation (nm)500
Emission (nm)522



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Authors: Ogura, Takeharu and Tanaka, Yoshiyuki and Tamaki, Hiroki and Harada, Mamoru
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NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo
Authors: Zhang, Lu and Han, Jianjun and Jackson, Am and a L , undefined and Clark, Leslie N and Kilgore, Joshua and Guo, Hui and Livingston, Nick and Batchelor, Kenneth and Yin, Yajie and Gilliam, Timothy P and others, undefined
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Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70
Authors: Howe, Matthew K and Bodoor, Khaldon and Carlson, David A and Hughes, Philip F and Alwarawrah, Yazan and Loiselle, David R and Jaeger, Alex M and Darr, David B and Jordan, Jamie L and Hunter, Lucas M and others, undefined
Journal: Chemistry & biology (2014): 1648--1659


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In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation
Authors: Lerma-Diaz JM, Hern and ez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno PC, Gomez-Contreras P, Cervantes-Munguia R, Scott-Algara D, Aguilar-Lemarroy A, Jave-Suarez LF, Bravo-Cuellar A.
Journal: Immunol Lett (2006): 149
Serofendic acid, a neuroprotective substance derived from fetal calf serum, inhibits mitochondrial membrane depolarization and caspase-3 activation
Authors: Kume T, Taguchi R, Katsuki H, Akao M, Sugimoto H, Kaneko S, Akaike A.
Journal: Eur J Pharmacol (2006): 69
Multiparameter measurement of caspase 3 activation and apoptotic cell death in NT2 neuronal precursor cells using high-content analysis
Authors: Fennell M, Chan H, Wood A.
Journal: J Biomol Screen (2006): 296
Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells
Authors: Jiang DJ, Jia SJ, Dai Z, Li YJ.
Journal: J Mol Cell Cardiol (2006): 529
Diallyl Trisulfide Induces Apoptosis of Human Gastric Cancer Cell Line MGC803 Through Caspase-3 Pathway.
Authors: Xiao XL, Peng J, Su Q, Xiang SL, Tang GH, Huang YS, Zhou XT.
Journal: Ai Zheng (2006): 1247
Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3
Authors: Miller TJ, Schneider RJ, Miller JA, Martin BP, Al-Ubaidi MR, Agarwal N, Dethloff LA, Philbert MA.
Journal: Neurotoxicology (2006): 44
Quantitative measurement of caspase-3 activity in a living starfish egg
Authors: Sakaue M, Motoyama Y, Yamamoto K, Shiba T, Teshima T, Chiba K.
Journal: Biochem Biophys Res Commun (2006): 878
Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe
Authors: Wu X, Simone J, Hewgill D, Siegel R, Lipsky PE, He L.
Journal: Cytometry A (2006): 477
Homogeneous, bioluminescent protease assays: caspase-3 as a model
Authors: O'Brien MA, Daily WJ, Hesselberth PE, Moravec RA, Scurria MA, Klaubert DH, Bulleit RF, Wood KV.
Journal: J Biomol Screen (2005): 137
Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro
Authors: Loop T, Dovi-Akue D, Frick M, Roesslein M, Egger L, Humar M, Hoetzel A, Schmidt R, Borner C, Pahl HL, Geiger KK, Pannen BH.
Journal: Anesthesiology (2005): 1147