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QIA72 Caspase-9 Activity Assay Kit, Fluorometric

Caspase-9 Activity Assay Kit, Fluorometric MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: ICE-LAP6 Assay Kit, APAF-3 Assay Kit, CASP9 Assay Kit, Mch6 Assay Kit

QIA72
  
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Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorescence
Description
Overview

This product has been discontinued.





Suitable for the in vitro detection of caspase-9 activity in cell lysates. The assay is simple, rapid, and includes all controls. Suitable for use in frozen samples. Assay is validated against the Cell Death Detection NMP ELISA Kit (Cat. No. QIA20).
Catalogue NumberQIA72
Brand Family Calbiochem®
SynonymsICE-LAP6 Assay Kit, APAF-3 Assay Kit, CASP9 Assay Kit, Mch6 Assay Kit
Materials Required but Not Delivered• 1 M dithiothreitol (DTT, Cleland reagent) solution (1.54 g to 10 ml H2O) made fresh (can be stored at -20°C for one month).
• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettes with disposable tips.
• Fluorescent plate reader capable of measuring excitation ~400 nm and emission at ~505 nm.
• A centrifuge, for spinning tubes or 96-well plate and to pulse-spin the Recombinant Caspase-9 vials.
• Microcentrifuge tubes.
• Protease Inhibitor Cocktail Set III (Cat. No. 539134)

Note: It is recommended to add protease inhibitor cocktail at 1:500 dilution to Sample Buffer and Assay Buffer just before use. These protease inhibitors have been shown to exhibit no effect on recombinant caspase-9 activity.
References
ReferencesX.Sun, M., et al. 1999. J. Biol. Chem. 274, 5053.
Thornberry, N.A. and Lazebnik, Y. 1998. Science 281, 1312.
Martin, S.J. and Green, D.R. 1995. Cell 82, 349.
Product Information
Detection methodFluorescence
Form96 Tests
Format96-well plate
Kit contains96-Well Plate, Caspase-9 Extraction Buffer, Assay Buffer, Recombinant Caspase-9, Caspase-9 Substrate Conjugate, Caspase-9 Inhibitor, Control Cell Pellets, and a user protocol.
Applications
Biological Information
Assay time2-3 h
Sample TypeCell lysate
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 22-36/37/38-41

Harmful if swallowed.
Irritating to eyes, respiratory system and skin.
Risk of serious damage to eyes.
S PhraseS: 23-26-36

Do not breathe fumes.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing.
Product Usage Statements
Intended useThe Calbiochem® Caspase-9 Activity Assay is a non-isotopic enzyme assay for the in vitro detection of caspase-9 activity in cell lysates. The assay can be adapted for 96-well plates for sensitive drug screening methods.
Storage and Shipping Information
Ship Code Multiple Storage Conditions
Toxicity Multiple Toxicity Values, refer to MSDS
Storage Multiple storage requirements
Storage ConditionsUpon arrival store Recombinant Caspase-9 (2 vials) at -70°C to maintain stability. Store all other kit components at -20°C. Once thawed, Caspase-9 Sample Buffer and Assay Buffer may be stored at 4°C. Do not expose reagents to excessive light.
Protect from Moisture Protect from moisture
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains96-Well Plate, Caspase-9 Extraction Buffer, Assay Buffer, Recombinant Caspase-9, Caspase-9 Substrate Conjugate, Caspase-9 Inhibitor, Control Cell Pellets, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number GTIN
QIA72 0

Documentation

Caspase-9 Activity Assay Kit, Fluorometric Certificates of Analysis

TitleLot Number
QIA72

References

Reference overview
X.Sun, M., et al. 1999. J. Biol. Chem. 274, 5053.
Thornberry, N.A. and Lazebnik, Y. 1998. Science 281, 1312.
Martin, S.J. and Green, D.R. 1995. Cell 82, 349.
User Protocol

Revision21-January-2008 JSW
SynonymsICE-LAP6 Assay Kit, APAF-3 Assay Kit, CASP9 Assay Kit, Mch6 Assay Kit
Form96 Tests
Format96-well plate
Detection methodFluorescence
Speciesa broad range of species
StorageUpon arrival store Recombinant Caspase-9 (2 vials) at -70°C to maintain stability. Store all other kit components at -20°C. Once thawed, Caspase-9 Sample Buffer and Assay Buffer may be stored at 4°C. Do not expose reagents to excessive light.
Intended useThe Calbiochem® Caspase-9 Activity Assay is a non-isotopic enzyme assay for the in vitro detection of caspase-9 activity in cell lysates. The assay can be adapted for 96-well plates for sensitive drug screening methods.
BackgroundCaspases are a family of cysteine proteases that act concertedly in a cascade triggered by apoptosis signaling. The culmination of this cascade is the cleavage of a number of proteins in the cell, followed by cell disassembly, cell death, and ultimately the phagocytosis and removal of the cell debris. Caspases do not engage in indiscriminate digestion of proteins during apoptosis, but rather a select set of proteins are targeted and cleaved at specific peptide sequences. The destruction of these proteins is genetically programmed and systematically carried out to ensure the demise of the cell. Caspases can be separated into two subsets based on whether they are at the initiating end of the apoptotic process (cell death signaling events), or at the effector end (cell disassembly). The initiator caspases, such as caspases-8 and -9, appear to activate other caspases at the effector end of the cascade, such as caspases-3, -6, and -7. It is the effector caspases that are largely responsible for the morpological and biochemical cell changes that are the hallmark of apoptosis. Poly (ADP-ribose) polymerase (PARP) is one select substrate of caspase-3. PARP is a component of the U1 small nuclear ribonucleoprotein and has important DNA repair capabilities. In humans, the caspase family consists of 14 proteolytic enzymes (caspases 1-14). The enzymes have in common similar cleavage specificities, amino acid sequences, and are expressed as proenzymes. Because of their similarities, caspases can be activated by each other or by autocatalysis. The individual caspases have different preferences for 4 amino-acid peptide recognition motifs in their substrates.
Principles of the assayThe Calbiochem® Caspase-9 Activity Assay detects caspase-9 activity in cell lysates. It takes advantage of the specificity of the enzyme for cleavage after aspartate residues in a particular peptide sequence (LEHD). To make detection possible, the LEHD substrate is labeled with a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC). Reaction can be monitored by a blue to green shift in fluorescence upon cleavage of the AFC fluorophore. We provide all the reagents necessary to lyse cells and extract caspase-9, if it is present in the sample.
Materials providedSufficient reagents have been provided to perform 100 tests. Two positive controls are provided with this assay, one of which can be treated with the specific Caspase-9 inhibitor (negative control). The controls, apoptotic HL-60 cells in a frozen cell pellet, are provided to ensure quality of the product. HL-60 cells have been cultured and treated with 0.5 µg/ml Actinomycin D for 19 hours to induce apoptosis. Caspase-9 is activated in response to the drug treatment. By comparing the signal obtained from a treated TEST sample to a untreated TEST sample, a ratio can be calculated to determine caspase-9 activity. Recombinant Caspase-9 is provided as an additional positive control.

To determine specificity of positive TEST samples, we have included a specific, cell-permeable, irreversible caspase-9 inhibitor (Z- LEHD -FMK). The N-terminal portion of this peptide allows cell permeability, while the C-terminal portion is a potent inhibitor of caspase-9 activity.

• 96-Well Plate (Kit Component No. JA1881): 1 plate
• Caspase-9 Sample Buffer (Kit Component No. JA1877): 1 bottle, 7 ml
• Assay Buffer (Kit Component No. JA1878): 1 bottle, 7 ml
• Recombinant Caspase-9 (Kit Component No. JA1889): 2 vials, 5 µl each
• Caspase-9 Substrate Conjugate (Kit Component No. JA1887): 1 bottle, 1.2 ml
• Caspase-9 Inhibitor (Kit Component No. JA1888): 1 vial, 20 µl
• Positive Control Cell Pellet (Kit Component No. JA1829): 2 Pellets
Materials Required but not provided• 1 M dithiothreitol (DTT, Cleland reagent) solution (1.54 g to 10 ml H2O) made fresh (can be stored at -20°C for one month).
• 2-20 µl, 20-200 µl and 200-1000 µl precision pipettes with disposable tips.
• Fluorescent plate reader capable of measuring excitation ~400 nm and emission at ~505 nm.
• A centrifuge, for spinning tubes or 96-well plate and to pulse-spin the Recombinant Caspase-9 vials.
• Microcentrifuge tubes.
• Protease Inhibitor Cocktail Set III (Cat. No. 539134)

Note: It is recommended to add protease inhibitor cocktail at 1:500 dilution to Sample Buffer and Assay Buffer just before use. These protease inhibitors have been shown to exhibit no effect on recombinant caspase-9 activity.
Precautions and recommendations• Do not expose reagents to excessive light.
• Wear disposable gloves and eye protection.
• Use only plates suitable for fluorometric assays.
• Do not mix reagents from different kits.
• Do not mouth pipette or ingest any of the reagents.
• The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products.
• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
• Human samples may be contaminated with infectious agents. Do not ingest, expose to open wounds, or breathe aerosols. Dispose of samples properly.
Detailed protocolAllow all reagents to reach room temperature before use with the exception of the Recombinant Caspase-9 and the positive control cell pellets which should be stored at -70°C or on dry ice and should be thawed immediately before use.

Protocol for Measuring Caspase-9 Activity in Cell Lysates (Tube):
Numerous extraction protocols can be used. The following protocol has been shown to work with a number of cell lines.

1. The recommended number of cells per test is 1 x 106 cells. For suspension cells, pellet by centrifugation at 500 x g for 5 min and remove supernatant (discard or test in Cell Death Detection (Nuclear Matrix Protein) ELISA Cat No. QIA20). For adherent cell lines, collect tissue culture medium into an appropriate centrifuge tube to be centrifuged with the attached cells. The tissue culture medium may contain detached cells that are dead or dying that may contain caspase-9. Add PBS to the culture flask and harvest cells by scraping. Pool with the tissue culture medium. Centrifuge at 500 x g for 5 min remove supernatant (discard or test in Cell Death Detection (Nuclear Matrix Protein) ELISA Cat No. QIA20).
2. The cell pellet can be tested immediately or frozen at -20°C and tested at a later date.
3. Remove the Positive Control Samples provided with the kit from -20°C. Two positive controls are provided with this assay, one of which can be treated with the specific caspase-9 inhibitor (negative control).
4. Calculate the amount of Sample Buffer needed based on 60 µl for every 1 X 106 cells (20% overage). Add 10 µl of the 1 M stock DTT solution for every 1 ml of Sample Buffer.
5. For every 1 X 106 cells, resuspend the sample pellets in 50 µl Sample Buffer and vortex. Add 50 µl Sample Buffer to each of the control cell pellets and vortex. From this point on, the control samples will be treated exactly as the unknown samples.
6. Incubate the samples on ice for 10 min.
7. Centrifuge the samples for 5 min at 500 x g.
8. Remove the plate from the packaging.
9. Transfer 50 µl cleared lysates to appropriate wells of the plate. Note, if you plan to add Caspase-9 Inhibitor to one or more of your samples, you will need two wells for these samples. As an additional control, pipette 50 µl Sample Buffer into one of the wells on the plate as a 'Buffer Control' or blank. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples. Extra sample should be aliquotted to avoid multiple freeze/thaws and frozen at -20°C to be tested at a later date if desired.
10. Transfer 50 µl of the prepared Positive and Negative Control lysates to appropriate wells of the plate.
11. Take one vial of Recombinant Caspase-9. Defrost by hand and then pulse-spin. Add 120 µl Sample Buffer directly to vial. Mix by pipetting. Transfer 50 µl Recombinant Caspase-9 to an apropriate well of the plate. Each recombinant enzyme vial has enough enzyme to do at least 2 reactions.
12. Calculate the amount of Assay Buffer needed based on 60 µl for every test well (20% overage). Add 10 µl of the 1 M stock DTT solution for every 1 ml of Assay Buffer.
13. Add 50 µl/well Assay Buffer to each assay well.
14. Pipet 1 µl of Caspase-9 Inhibitor into one control lysate well (negative control). If you choose to use the Caspase-9 Inhibitor as a specificity control of a test sample, pipet 1 µl of Caspase-9 Inhibitor into the appropriate wells at this point. Cover the plate with its cover and incubate at 37°C for 30 min. If you are NOT using the Caspase-9 Inhibitor, proceed directly to step 15.
15. Add 10 µl/well Caspase-9 Substrate Conjugate.
16. As an additional control read the plate immediately after addition of the substrate using a fluorescent plate reader capable of measuring excitation ~400 nm, and emission at ~505 nm. The data can then be represented as the delta or difference between zero time and one to 2 h.
17. Cover the plate and incubate at 37°C for 1 to 2 h (there is an increase in sensitivity if a 2 h incubation is used).
18. Read the plate using a fluorescent plate reader capable of measuring excitation ~400 nm, and emission at ~505 nm. The assay was validated with a BMG FLUOstar fluorometer with a 390 nm excitation and a 510 nm emission filter.

Protocol for Measuring Caspase-9 Activity in Cell Lysates (96-Well Plate):

1. Grow cells in desired media to a density of about 0.5-1 x 106 cells/well in a sterile 96-well tissue culture plate suitable for reading using a fluorimeter (for example, a black plate with clear bottom and sterile for tissue culture). Leave enough wells for the appropriate controls. Note, if you plan to add Caspase-9 Inhibitor to one or more of your samples, you will need one extra well for each sample. Treat cells with drug(s) of choice at the appropriate dose and time courses.
2. Spin the 96-well plate at 500 x g for 5 min. Remove the supernatant by emptying contents into a reservoir.
3. Calculate the amount of Sample Buffer needed based on 60 µl for every test well (20% overage). Add 10 µl of the 1 M stock DTT solution for every 1 ml of Sample Buffer.
4. Add 50 µl/well Sample Buffer. Pipette the liquid in each well several times to ensure the best extraction possible. Incubate the plate at 4°C for 30 min.
5. Remove the Positive Control provided with the kit from -20°C. Two positive controls are provided with this assay, one of which can be treated with the specific caspase-9 inhibitor (negative control). Add 50 µl Sample Buffer to each of the control cell pellets and mix gently. Incubate the samples on ice for 10 min. Centrifuge the samples for 5 min at 500 x g. Transfer 50 µl of cleared control lysates to appropriate wells of the plate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.
6. As an additional control, pipette 50 µl Sample Buffer into one of the wells on the plate as a 'Buffer Control' or blank.
7. Take 1 vial of Recombinant Caspase-9. Defrost by hand and then pulse-spin. Add 120 µl Sample Buffer directly to vial. Mix by pipetting. Transfer the 50 µl of Recombinant Caspase-9 to an appropriate well of the plate. Each recombinant enzyme vial has enough enzyme to do at least 2 reactions.
8. Calculate the amount of Assay Buffer needed based on 60 µl for every test well (20% overage). Add 10 µl of the 1 M stock DTT solution for every 1 ml of Assay Buffer.
9. Add 50 µl/well Assay Buffer to each assay well.
10. Pipet 1 µl of Caspase Inhibitor into one control lysate well (negative control). If you choose to use the Caspase-9 Inhibitor as a specificity control of a test sample, pipet 1 µl of Caspase-9 Inhibitor into the appropriate wells at this point. Cover the plate and incubate at 37°C for 30 min. If you are NOT using the Caspase-9 Inhibitor, proceed directly to step 11.
11. Add 10 µl Caspase-9 Fluorescent Substrate Conjugate to each well.
12. As an additional control read the plate immediately after addition of the substrate using a fluorescent plate reader capable of measuring excitation ~400 nm and emission at ~505 nm. The data can then be represented as the delta or difference between zero time and one to two hours.
13. Cover the plate and incubate at 37°C for 1-2 h (there is an increase in sensitivity if a 2 h incubation is used).
14. Read the plate using a fluorescent plate reader capable of measuring excitation ~400 nm and emission at ~505 nm. The assay was validated with a BMG FLUOstar fluorometer with a 390 nm excitation and a 510 nm emission filter.
CalculationsThe data can be expressed as the relative signal (RFU) after subtraction of the relative signal of the appropriate buffer controls. The data may also be expressed as a ratio of signal obtained from a treated sample to an untreated control sample after subtraction of the appropriate buffer controls. The ratio of the signal of treated to untreated sample is reasonably constant over a wide range of cell concentrations. In addition, as stated above the data may be expressed as the difference between readings at two different time points such as at zero time and 2 h after the addition of substrate.
Assay characteristics and examplesThe Caspase-9 Activity Assay detects apoptosis in many different tissue culture cells induced by many different agents and physiological conditions (Figures 1-8). Specificity for apoptotic cell death has been demonstrated when caspase activity has been compared to results
obtained from standard apoptotic assays such as the Nucleosome ELISA Kit (Cat No. QIA20). Inhibition studies (see Figure 7 for a typical
study) demonstrate specificity. The number of cells over a broad range affects the signal but not the ability to detect apoptosis.

Figure 1: Correspondence of Caspase Activity of A-431 Cells

Correspondence of caspase activity of A-431, an epidermoid carcinoma cell line, with apoptosis. A-431 cells (5 X 105 cells/ml) that had been growing for 18 h (37°C and 7% CO2) were treated with 1 µM Dexamethasone (Dex) for 48 h, 0.5 µg/ml Actinomycin D (ActD) for 24 h or not treated (Untrx). Following drug treatment the detached apoptotic cells present in the tissue culture supernatant were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity Assay and the Nucleosome ELISA. The supernatants were tested for apoptosis induced release of nuclear matrix protein with the Cell Death Detection ELISA. The lower levels of apoptosis in the Dexamethasone treated A431 cells detected by the very sensitive NMP and Nucleosome ELISAs correspond to lack of caspase activity. The high levels of cell death detected by the two apoptotic assays in the Actinomycin D treated samples correspond to the high levels of caspase activity. The attached cell populations were also harvested by scraping and analyzed. The level of Caspase-9 activity was about 15% of the detached populations (data not shown).


Figure 2: Correspondence of Caspase Activity of SW620 Cells

Correspondence of caspase activity of SW620, a colorectal adenocarcinoma cell line, with apoptosis. SW620 cells (5 X 105 cells/ml) that had been growing for 18 h (37°C and 7% CO2) were treated with one of the following: 0.5 µM Staurosporine (Stauro) and 3 µM Camptothecin (Camp) for 24 h, and 1 µM Dexamethasone (Dex), 1 µg/ml Actinomycin D (ActD), and 1 nM Taxol, for 48 h, or not treated (Untrx).Following drug treatment the detached apoptotic cells present in the tissue culture supernatant were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity Assay and the Nucleosome ELISA. The supernatants were tested for apoptosis induced release of nuclear matrix protein with the Cell Death Detection ELISA. The attached cell populations were also harvested by scraping and analyzed The levels of apoptosis detected by the NMP assay generally correspond to the caspase activity of the detached cell population, except exposure to camptothecin resulted in more Caspase-9 activity than taxol or dexamethasone for the amount of apoptosis. Analysis of the attached cells indicates that staurosporine drug treated samples had significantly more Caspase-9 activity than the untreated control population.


Figure 3: Correspondence of Caspase-9 Activity of HT-1080 Cells

Correspondence of Caspase-9 activity of HT-1080, a fibrosarcoma cell line, with apoptosis. HT-1080 cells (5 X 105 cells/ml) that had been growing for 18 h (370C and 7% CO2) were treated with one of the following: 1 µM Dexamethasone (Dex) for 48 h, 3 µM Camptoththecin (Camp) for 24 h, 0.5 µM Stauroporine (Stauro) for 24 h or not treated (Untrx). Following drug treatment the detached apoptotic cells present in the tissue culture supernatant were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity Assay and the Nucleosome ELISA. The supernatants were tested for apoptosis induced release of nuclear matrix protein with the Cell Death Detection ELISA. The low levels of apoptosis in the dexamethasome induced detached cell correlated to low levels of Caspase-9 activity and the high levels of apoptosis in the camptothecin and staurosporine induced detachment correlated to high levels of Caspase-9 activity. The attached cell populations were also harvested by scraping and analyzed. The drug treated attached populations showed a similar trend of a low level of Caspase-9 activity in the dexmethasome treated cells and a high level of Caspase-9 activity in the camptothecin and staurosporine treated cells (data not shown).














Figure 4: Correspondence of Caspase Activity of HL-60 Cells

Correspondence of caspase activity of HL-60, an acute promyelocytic leukemia cell line, with Camptothecin induced apoptosis. HL-60 cells (8 X 105 cells/ml) were treated with 3 µM Camptothecin (Camp) for 24 h or not treated (Untrx). Following drug treatment the cells were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity Assay and the Nucleosome ELISA. The high level of apoptosis detected by the nucleosome assay correlated to high levels of Caspase-9 activity


Figure 5: Correspondence of Caspase-9 Activity of HL-60 Cells

Correspondence of Caspase-9 activity of HL-60 Actinomycin D induced apoptosis. HL-60 cells (8 X 105 cells/ml) were treated with 0.5 µg/ml Actinomycin D (ActD) for 24 h or not treated (Untrx). Following drug treatment the cells were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity Assay and the Nucleosome ELISA. The supernatants were tested for apoptosis induced release of nuclear matrix protein with the Cell Death Detection ELISA. The high level of apoptosis detected by the nucleosome and the NMP assays correlated to high levels of Caspase-9 activity.


Figure 6: Correspondence of Caspase-9 Activity of Jurkat Cells

Correspondence of Caspase-9 activity of Jurkat, an acute T cell leukemia cell line, with Actinomycin D induced apoptosis. Jurkat cells (1 X 106 cells/ml) were treated with 0.5 µg/ml Actinomycin D (Act D) for 24 h or not treated (Untrx). Following drug treatment the cells were harvested by slow speed centrifugation and analyzed in the Caspase-9 Activity and the Nucleosome ELISA. The supernatants were tested for apoptosis induced release of nuclear matrix protein with the Cell Death Detection ELISA. The high level of apoptosis detected by the nucleosome and the NMP assays correlated to high levels of Caspase-9 activity.











Figure 7: Specificity of the signal

The signals of the drug treated samples in the presence of a specific inhibitor of Caspase-9 are very similar and very low. This is consistent with the concept that most of the signal is due to Caspase-9 activity.


Figure 8: Caspase-9 Assay

Freezing and thawing has only a slight effect on the signal of a lysate and on the ratio between treated and untreated samples.
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