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

Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 uL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 uM for 5 hours while the untreated cells were used as control. The caspase 3/7 assay solution (100 uL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm with FlexStation fluorescence microplate reader (Molecular Devices).
Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 uL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 uM for 5 hours while the untreated cells were used as control. The caspase 3/7 assay solution (100 uL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm with FlexStation fluorescence microplate reader (Molecular Devices).
Detection of Caspase 3/7 Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/90 uL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 uM for 5 hours while the untreated cells were used as control. The caspase 3/7 assay solution (100 uL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 540/620 nm with FlexStation fluorescence microplate reader (Molecular Devices).
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Spectral properties
Excitation (nm)532
Emission (nm)619
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Excitation (nm)
532
Emission (nm)
619
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.

Platform


Fluorescence microplate reader

Excitation535 nm
Emission620 nm
Cutoff610 nm
Recommended plateSolid black

Components


Example protocol


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.

PREPARATION OF STOCK SOLUTION

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.

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.

Spectrum


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Spectral properties

Excitation (nm)532
Emission (nm)619

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Citations


View all 16 citations: Citation Explorer
Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells
Authors: Takakura, Masatoshi and Mizutani, Ayano and Kudo, Mizuki and Ishikawa, Airi and Okamoto, Takuya and Fu, Tong Xuan and Kurimoto, Shin-ichiro and Koike, Yuka and Mishima, Kenji and Tanaka, Junichi and others,
Journal: Biological and Pharmaceutical Bulletin (2024): 138--144
Respiratory syncytial virus--approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors
Authors: Ettich, Julia and Wittich, Christoph and Moll, Jens M and Behnke, Kristina and Floss, Doreen M and Reiners, Jens and Christmann, Andreas and Lang, Philipp A and Smits, Sander HJ and Kolmar, Harald and others,
Journal: The Journal of Biological Chemistry (2023)
Respiratory syncytial virus (RSV)-approved monoclonal antibody Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors
Authors: Ettich, Julia and Wittich, Christoph and Moll, Jens M and Behnke, Kristina and Floss, Doreen M and Reiners, Jens and Christmann, Andreas and Lang, Philipp A and Smits, Sander HJ and Kolmar, Harald and others,
Journal: Journal of Biological Chemistry (2023): 105270
Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer
Authors: Salvati, Annamaria and Melone, Viola and Sellitto, Assunta and Rizzo, Francesca and Tarallo, Roberta and Nyman, Tuula A and Giurato, Giorgio and Nassa, Giovanni and Weisz, Alessandro
Journal: Breast Cancer Research (2022): 1--23
Design and evaluation of folate-modified liposomes for pulmonary administration in lung cancer therapy
Authors: Onodera, Risako and Morioka, Shunsuke and Unida, Shinshu and Motoyama, Keiichi and Tahara, Kohei and Takeuchi, Hirofumi
Journal: European Journal of Pharmaceutical Sciences (2022): 106081
Intracellular accumulation of advanced glycation end products induces osteoblast apoptosis via endoplasmic reticulum stress
Authors: Suzuki, Ryusuke and Fujiwara, Yukio and Saito, Mitsuru and Arakawa, Shoutaro and Shirakawa, Jun-ichi and Yamanaka, Mikihiro and Komohara, Yoshihiro and Marumo, Keishi and Nagai, Ryoji
Journal: Journal of bone and mineral research (2020): 1992--2003
Helicobacter pylori secreted protein HP1286 triggers apoptosis in macrophages via TNF-independent and ERK MAPK-dependent pathways
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: Frontiers in Cellular and Infection Microbiology (2017): 58
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)
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,
Journal: British journal of cancer (2017): 1177--1185

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