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Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*

Detection of caspase 9 activities using Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit in Jurkat cells. Jurkat cells were seeded at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with 10 mM adenosine for 48 hours while the untreated cells were used as control. The caspase 9 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 with a FlexStation™ microplate reader (Molecular Devices).
Detection of caspase 9 activities using Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit in Jurkat cells. Jurkat cells were seeded at 200,000 cells/90 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with 10 mM adenosine for 48 hours while the untreated cells were used as control. The caspase 9 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 with a FlexStation™ microplate reader (Molecular Devices).
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Catalog Number22799
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. This particular kit is designed to monitor cell apoptosis by measuring caspase 9 activity. Caspase 9 is a member of the CED-3 subfamily. Activated Caspase-9 cleaves downstream caspases such as caspase-3, -6 and -7, initiating the caspase cascade. It is essential for apoptosis during normal development of the central nervous system. Caspase 9 is proven to have selectivity for the peptide sequence Leu-Glu-His-Asp (LEHD). This kit uses (Ac-LEHD)2-R110 as a fluorogenic indicator for caspase 9 activity. Cleavage of R110 peptides by caspase 9 generates strongly fluorescent rhodamine 110 (R110)which is monitored at the emission between 520 nm and 530 nm with the excitation between 480 nm and 500 nm. The kit provides all the essential components. The assay is robust and can be readily adapted for high throughput screening. It can be used to either quantify the activated caspase 9 activities in apoptotic cells or screen the caspase 9 inhibitors. Quite a few labs have used this kit for high throughput screenings.

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 9 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
  2. Add equal volume of Caspase 9 Substrate working solution
  3. Incubate at room temperature for 30 min to 1 hour
  4. Monitor the fluorescence at Ex/Em = 490/525 nm

Important notes
Thaw all the kit components to room temperature before use.

PREPARATION OF WORKING SOLUTION

Add 50 μL of Caspase 9 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix them well. Note: Aliquot and store the unused Caspase 9 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 to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 9 Substrate working solution.

  4. Incubate the working solution plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-LEHD-CHO caspase 9 inhibitor to selected samples 10 minutes before adding the assay working solution at room temperature to confirm the inhibition of the caspase 9-like activity.

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

  6. Monitor the fluorescence intensity 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 2 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)
The BH3 mimetic ($\pm$) gossypol induces ROS-independent apoptosis and mitochondrial dysfunction in human A375 melanoma cells in vitro
Authors: Haasler, Lisa and Kondadi, Arun Kumar and Tsigaras, Thanos and von Montfort, Claudia and Graf, Peter and Stahl, Wilhelm and Brenneisen, Peter
Journal: Archives of Toxicology (2021): 1349--1365

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
Authors: Chun W, Waldo GS, Johnson GV.
Journal: J Neurochem (2007): 2529