Amplite® Colorimetric Caspase 3/7 Assay Kit Yellow Color

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.

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

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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

Citations

View all 11 citations: Citation Explorer

GENERATION OF Usp16 AND Bmi1 KNOCKOUT BREAST CANCER CELL LINES BY CRISPR/Cas9 TECHNOLOGY
Authors: Garland, Danielle C
Journal: (2024)

Linking pesticide exposure to neurodegenerative diseases: An in vitro investigation with human neuroblastoma cells
Authors: Alehashem, Maryam and Alcaraz, Alper James and Hogan, Natacha and Weber, Lynn and Siciliano, SD and Hecker, M
Journal: Science of The Total Environment (2024): 173041

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)

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

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

References

View all 67 references: Citation Explorer

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

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

Page updated on November 20, 2024

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