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

Detection of caspase 3/7 Activity in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a black wall/clear bottom 96-well costar plate. The cells were treated with or without 20 µM ofcamptothecin for 5 hours, and/or 5 µM caspase 3/7 inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assaysolution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity wasmeasured at Ex/Em = 490/525 nm using the NOVOstar instrument (BMG Labtech).
Detection of caspase 3/7 Activity in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a black wall/clear bottom 96-well costar plate. The cells were treated with or without 20 µM ofcamptothecin for 5 hours, and/or 5 µM caspase 3/7 inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assaysolution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity wasmeasured at Ex/Em = 490/525 nm using the NOVOstar instrument (BMG Labtech).
Detection of caspase 3/7 Activity in Jurkat cells. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a black wall/clear bottom 96-well costar plate. The cells were treated with or without 20 µM ofcamptothecin for 5 hours, and/or 5 µM caspase 3/7 inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assaysolution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity wasmeasured at Ex/Em = 490/525 nm using the NOVOstar instrument (BMG Labtech).
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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
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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 (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)2R110 as fluorogenic indicator for assaying caspase-3 activity. Cleavage of R110 peptides by caspases generates strongly fluorescent R110 that can be monitored fluorimetrically at 510-530 nm with excitation of 488 nm, the most common excitation light source used in fluorescence instruments. R110-derived caspase substrates are probably the most sensitive indicators widely used for the fluorimetric detection of various caspase activities. 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

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

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 hour
  4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important notes
Thaw component A, B, C (if desired, Component D and E) at room temperature before starting the experiment.

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. Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO (not provided) directly to the vial of Ac-DEVD-CHO (Component D), and mix well to make 1 mM Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution. This Caspase 3/7 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 Caspase 3/7 Substrate stock solution (Component A) and 100 μL of 1M DTT solution (Component C) into 10 mL of 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. Protect from light.

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

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells with 10 µL of 10X test compound (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 desired period of time ( 4 - 6 hours for Jurkat cells treated with camptothecin) 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, protected from light. Note: If desired, add 1 µL of 1 mM Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the Caspase 3/7-like activities.  Note: If desired, prepare a R110 standards by diluting 5 mM  R110 Standard (Component E) into growth Medium to yield serially diluted R110 standards ranging from 0 - 50 µM. Add 100 µL of the serially diluted R110 standards into the wells containing 100 µL of Caspase 3/7 working solution at any time prior to measuring the fluorescence. This standard curve could be used to determine the moles of product produced in the caspase 3/7 containing reactions.

  5. Centrifuge cell plates (especially for the non-adherent cells) at 800 rpm for 2 minutes with brake off. 

  6. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm).

Spectrum


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spectrum

Spectral properties

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

Images


Citations


View all 22 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
Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95
Authors: Minafra, Anna Rita and Rafii, Puyan and Mossner, Sofie and Bazgir, Farhad and Floss, Doreen M and Moll, Jens M and Scheller, J{\"u}rgen
Journal: Journal of Biological Chemistry (2023): 104989
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
Apoptosis induction by extracorporeal photopheresis is enhanced by increasing the 8-methoxypsoralen concentration and by replacing plasma with saline
Authors: H{\"a}hnel, Viola and Brosig, Andreas-Michael and Ehrenschwender, Martin and Burkhardt, Ralph and Offner, Robert and Ahrens, Norbert
Journal: Transfusion (2021)
Pericentromeric Satellite III transcripts induce etoposide resistance
Authors: Kanne, Julian and Hussong, Michelle and Isensee, J{\"o}rg and Mu{\~n}oz-L{\'o}pez, {\'A}lvaro and Wolffgramm, Jan and He{\ss}, Felix and Grimm, Christina and Bessonov, Sergey and Meder, Lydia and Wang, Jie and others,
Journal: Cell Death \& Disease (2021): 1--15
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
Senotherapeutic drugs for human intervertebral disc degeneration and low back pain
Authors: Cherif, Hosni and Bisson, Daniel G and Mannarino, Matthew and Rabau, Oded and Ouellet, Jean A and Haglund, Lisbet
Journal: Elife (2020): e54693

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