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Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*

Detection of caspase 8 activities in Jurkat cells using Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 20 µM of camptothecin for 4 hours. The caspase 8 Substrate working solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) with a FlexStation™ microplate reader (Molecular Devices).
Detection of caspase 8 activities in Jurkat cells using Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 20 µM of camptothecin for 4 hours. The caspase 8 Substrate working solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) with a FlexStation™ microplate reader (Molecular Devices).
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Catalog Number22798
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
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
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OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
80000
Excitation (nm)
500
Emission (nm)
522
Our Cell Meter™ assay kits are a set of tools for monitoring cellular functions. There are a variety of parameters that can be used to monitor cell apoptosis. This particular kit is designed to monitor cell apoptosis by measuring caspase 8 activity. Caspase 8 is a caspase protein, encoded by the CASP8 gene. Caspase 8 also plays an important role in neurodegenerative diseases such as Huntington disease. Caspase 8 is proven to have substrate selectivity for the peptide sequence Ile-Glu-Thr-Asp (IETD). This kit uses (Ac-IETD)2-R110 as a fluorogenic indicator for caspase 8 activity. Cleavage of rhodamine 110 (R110) peptides by caspase 8 generates strongly fluorescent R110 which is monitored at the emission between 520 nm and 530 nm with the excitation between 480 nm and 500 nm. This spectral feature makes the kit compatible with the FITC filter set. The kit provides all the essential components with an optimized assay protocol. The assay can be readily adapted for high throughput screenings. It can be used to either quantify the activated caspase 8 activities in apoptotic cells or screen the caspase 8 inhibitors.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Top/Bottom read mode

Components


Component A: Caspase 8 Substrate2 vials (50 µL/vial)
Component B: Assay Buffer1 bottle (20 mL)

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 8 Substrate working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
  3. Incubate at room temperature for 30 min to 1 hour
  4. Monitor fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important notes
Thaw one vial of each kit component at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 50 µL of Caspase 8 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspasae 8 Substrate working solution is not stable, use it promptly. Note: Aliquot and store unused Caspase 8 Substrate (Component A) and Assay Buffer (Component B) at -20 oC. Avoid repeated freeze/thaw cycles.

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/well of 10X test compounds (96-well plate) or 5 µL/well of 5X test compounds (384-well plate) into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.

  2. Incubate the cell plate in a 5% CO2, 37°C incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin or staurosporine) to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 8 Substarte working solution.

  4. Incubate the plate at room temperature for 30 min to 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-IETD-CHO caspase 8 inhibitor to selected samples 10 minutes before adding Caspase 8 Substrate working solution at room temperature to confirm the inhibition of the caspase 8-like activities.

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

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

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

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

Citations


View all 18 citations: Citation Explorer
Combination of Goniothalamin and Sol-Gel-Derived Bioactive Glass 45S5 Enhances Growth Inhibitory Activity via Apoptosis Induction and Cell Cycle Arrest in Breast Cancer Cells MCF-7
Authors: Bakar, Siti Aishah Abu and Ali, Abdul Manaf and Noor, Siti Noor Fazliah Mohd and Hamid, Shahrul Bariyah Sahul and Azhar, Nur Asna and Mohamad, Noor Muzamil and Ahmad, Nor Hazwani
Journal: BioMed Research International (2022)
RPL21 siRNA blocks proliferation in pancreatic cancer cells by inhibiting DNA replication and inducing G1 arrest and apoptosis
Authors: Li, Chaodong and Ge, Mei and Chen, Daijie and Sun, Tao and Jiang, Hua and Xie, Yueqing and Lu, Huili and Zhang, Baohong and Han, Lei and Chen, Junsheng and others,
Journal: Frontiers in oncology (2020): 1730
CD5L-induced activation of autophagy is associated with hepatoprotection in ischemic reperfusion injury via the CD36/ATG7 axis
Authors: Li, Junjian and Lin, Wei and Zhuang, Lei
Journal: Experimental and Therapeutic Medicine (2020): 2588--2596
Promotion of osteointegration under diabetic conditions by tantalum coating-based surface modification on 3-dimensional printed porous titanium implants
Authors: Wang, Lin and Hu, Xiaofan and Ma, Xiangyu and Ma, Zhensheng and Zhang, Yang and Lu, Yizhao and Li, Xiang and Lei, Wei and Feng, Yafei
Journal: Colloids and Surfaces B: Biointerfaces (2016): 440--452
microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2
Authors: He, Wei and Feng, Jianfang and Zhang, Yan and Wang, Yuanyuan and Zang, Wenqiao and Zhao, Guoqiang
Journal: Laboratory Investigation (2016): 317--324
Hypoxia regulates TRAIL sensitivity of colorectal cancer cells through mitochondrial autophagy.
Authors: Knoll, Gertrud and Bittner, Sebastian and Kurz, Maria and Jantsch, Jonathan and Ehrenschwender, Martin
Journal: Oncotarget (2016)
Role of delta-like ligand-4 in chemoresistance against docetaxel in MCF-7 cells
Authors: Wang, Q and Shi, Y and Butler, HJ and Xue, J and Wang, G and Duan, P and Zheng, H
Journal: Human & Experimental Toxicology (2016): 0960327116650006
Angiopoietins Modulate Survival, Migration, and the Components of the Ang-Tie2 Pathway of Chronic Lymphocytic Leukaemia (CLL) Cells In Vitro
Authors: Palma, Luis Mario Aguirre and Flamme, Hanna and Gerke, Iris and Kreuzer, Karl-Anton
Journal: Cancer Microenvironment (2016): 13--26
t-BHQ Provides Protection against Lead Neurotoxicity via Nrf2/HO-1 Pathway
Authors: Ye, Fang and Li, Xiaoyi and Li, Lili and Yuan, Jing and Chen, Jun
Journal: Oxidative medicine and cellular longevity (2015)

References


View all 31 references: Citation Explorer
Discovery of substituted N'-(2-oxoindolin-3-ylidene)benzohydrazides as new apoptosis inducers using a cell- and caspase-based HTS assay
Authors: Sirisoma N, Pervin A, Drewe J, Tseng B, Cai SX.
Journal: Bioorg Med Chem Lett (2009): 2710
Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: Structure-activity relationships of the 4-, 5-, 6-, 7- and 8-positions
Authors: Kemnitzer W, Kuemmerle J, Jiang S, Sirisoma N, Kasibhatla S, Crogan-Grundy C, Tseng B, Drewe J, Cai SX.
Journal: Bioorg Med Chem Lett (2009): 3481
Selective identification of cooperatively binding fragments in a high-throughput ligation assay enables development of a picomolar caspase-3 inhibitor
Authors: Schmidt MF, El-Dahshan A, Keller S, Rademann J.
Journal: Angew Chem Int Ed Engl (2009): 6346
Modified caspase-3 assay indicates correlation of caspase-3 activity with immunity of nonhuman primates to Yersinia pestis infection
Authors: Welkos S, Norris S, Adamovicz J.
Journal: Clin Vaccine Immunol (2008): 1134
Liver apoptosis following normothermic ischemia-reperfusion: in vivo evaluation of caspase activity by FLIVO assay in rats
Authors: Cursio R, Colosetti P, Auberger P, Gugenheim J.
Journal: Transplant Proc (2008): 2038
Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 4. Structure-activity relationships of N-alkyl substituted pyrrole fused at the 7,8-positions
Authors: Kemnitzer W, Drewe J, Jiang S, Zhang H, Crogan-Grundy C, Labreque D, Bubenick M, Attardo G, Denis R, Lamothe S, Gourdeau H, Tseng B, Kasibhatla S, Cai SX.
Journal: J Med Chem (2008): 417
Discovery of (naphthalen-4-yl)(phenyl)methanones and N-methyl-N-phenylnaphthalen-1-amines as new apoptosis inducers using a cell- and caspase-based HTS assay
Authors: Jiang S, Crogan-Grundy C, Drewe J, Tseng B, Cai SX.
Journal: Bioorg Med Chem Lett (2008): 5725
A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors
Authors: Valanne A, Malmi P, Appelblom H, Niemela P, Soukka T.
Journal: Anal Biochem (2008): 71
Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro -1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay
Authors: Drewe J, Kasibhatla S, Tseng B, Shelton E, Sper and io D, Yee RM, Litvak J, Sendzik M, Spencer JR, Cai SX.
Journal: Bioorg Med Chem Lett (2007): 4987
Split GFP complementation assay: a novel approach to quantitatively measure aggregation of tau in situ: effects of GSK3beta activation and caspase 3 cleavage
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Journal: J Neurochem (2007): 2529