Amplite® Colorimetric Caspase 3/7 Assay Kit Yellow Color

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Spectral properties

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

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Extinction coefficient (cm ^-1 M ^-1)

80000

Excitation (nm)

500

Emission (nm)

522

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.

Platform

Absorbance microplate reader

Absorbance	490 nm
Recommended plate	Clear bottom

Components

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 working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
Incubate at room temperature for 1 - 2 hours
Monitor absorbance at 490 nm

CELL PREPARATION

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

PREPARATION OF WORKING SOLUTION

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.

SAMPLE EXPERIMENTAL PROTOCOL

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.
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.
Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 working solution.
Incubate the plate at room temperature for at least 1 hour, protected from light.
Centrifuge cell plates at 800 rpm for 2 minutes with brake off.
Monitor the absorbance increase with an absorbance plate reader at OD =490 nm.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

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

Product Family

Name	Excitation (nm)	Emission (nm)
Amplite® Fluorimetric Caspase 3/7 Assay Kit Blue Fluorescence	341	441
Amplite® Fluorimetric Caspase 3/7 Assay Kit Green Fluorescence	500	522
Amplite® Fluorimetric Caspase 3/7 Assay Kit Red Fluorescence	532	619

Images

Figure 1. 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).

Citations

View all 9 citations: Citation Explorer

Chrysin Mediates the Induction of Apoptosis in Breast Cancer Cells via the Inhibition of the WNT/$\beta$-Catenin Signaling Pathway
Authors: {\c{C}}etinkaya, S{\"u}meyra
Journal: (2023)

Cross-Linked Cytochrome c-Based Nanoparticles for Targeted and Controlled Cancer Therapy
Authors: Martinez, Irivette Dominguez
Journal: (2022)

Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC)
Authors: Dominguez-Martinez, Irivette and Joaquin-Ovalle, Freisa and Ferrer-Acosta, Yancy and Griebenow, Kai H
Journal: Pharmaceutics (2022): 490

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

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

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

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

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
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: 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

References

View all 67 references: Citation Explorer

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

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

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

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

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

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

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

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

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

Caspase-3 activation and induction of PARP cleavage by cyclic dipeptide cyclo(Phe-Pro) in HT-29 cells
Authors: Brauns SC, Dealtry G, Milne P, Naude R, Van de Venter M.
Journal: Anticancer Res (2005): 4197