ApoAlert® Caspase Assay Plates
User Manual
PT3671-1 (PR24952)
Published 19 April 2002
ApoAlert® Caspase Assay Plates User Manual
Table of Contents
I. Introduction
3
II. List of Components
6
III. Additional Materials Required 6
IV. Caspase Assay Plate Protocol
7
A. General Considerations
7
B. Assay Procedure
8
V. References
9
VI. Related Products
10
List of Figures
Figure 1. ApoAlert® Caspase Profiling Plate
Figure 2. Detecting protease activity using
ApoAlert®
5
Caspase Assay Plates5
Notice to Purchaser
This product is intended to be used for research purposes only. It is not to be used for drug or diagnostic
purposes, nor is it intended for human use. Clontech products may not be resold, modified for resale, or
used to manufacture commercial products without written approval of Clontech Laboratories, Inc.
Parafilm® is a registered trademark of American National Can Co.
ApoAlert® is a registered trademark of Clontech Laboratories, Inc.
Clontech, Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc.
Clontech is a Takara Bio Company. ©2005
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Protocol # PT3671-1
Version # PR24952
ApoAlert® Caspase Assay Plates User Manual
I. Introduction
The ApoAlert® Caspase Assay Plates contain the fluorogenic substrates
specific for different caspases immobilized in the wells of a 96-well plate. When
cell lysate containing the active caspase is applied to the wells, the caspase will
cleave its substrate and a fluorescent product is released that can be detected
with a standard fluorescence plate reader.
Caspase Assay Plates enable high-throughput analysis of the apoptotic caspase
response. This assay design is ideal for studies involving multiple cell types,
or multiple cell treatments. These plates are provided in two formats: a single
caspase format for studies that focus on a specific caspase, or a profiling
format for analyzing several different caspases simultaneously (caspase-3, -8,
-9 & -2; Figure 1).
Background
Caspases are part of a large family of cysteine proteases that mediate programmed
cell death (apoptosis). Upon activation, they disable cellular housekeeping and
repair programs and cleave important structural components in the cell, causing
the morphological and functional changes characteristic of apoptotic cells.
Caspases are synthesized in the cytosol of mammalian cells as inactive zymogens,
which become active through intracellular caspase cascades (Cohen, 1997).
ApoAlert Caspase Assay Plates allow you to detect the activity of one or several
specific caspases, including: caspase-2, caspase-3, caspase-8, or caspase9—from cellular extracts in a 96-well format.
Caspase-2 is one of the first caspases to be identified, but little is known of its
mechanism for inducing apoptosis. It is a downstream caspase that seems to be
dependent upon caspase-3 activity (Paroni et al., 2001). Recent data suggests
that caspase-2 may act as a direct effector of the mitochondrial apoptotic pathway
(Guo et al., 2002).
Caspase-3 is involved in the execution phase of apoptosis, where cells undergo
morphological changes such as DNA fragmentation, chromatin condensation, and
apoptotic body formation (Porter & Janicke, 1999; Zou et al., 1999). Caspase-3
is activated in response to serum withdrawal, activation of the Fas cell surface
receptor cascade, treatment with radiation or pharmacological agents (Zou et
al., 1999).
Caspase-8, the first caspase in the CD95 apoptotic pathway, is activated by the
signalling pathways for CD95/Fas and tissue necrosis factor (TNF; reviewed in
Nicholson & Thornberry, 1997; Thornberry & Littlewood, 1998; Fraser & Evan,
1996; Cohen, 1997). Upon activation by cell-surface receptors, such as Fas,
caspase-8 directly or indirectly initiates the proteolytic activities of downstream
effector caspases, such as caspase-3 (Srinivasula et al., 1996; Muzio et al.,
1997; Zou et al., 1999).
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ApoAlert® Caspase Assay Plates User Manual
I. Introduction continued
Caspase-9, another upstream caspase, is activated via the mitochondrial release
of cytochrome c to the cytosol. Released cytochrome c binds to the apoptotic
protease activating factor (Apaf-1) in the cytosol, where it forms a complex that
activates procaspase-9 (Zou et al., 1999; Cain et al., 1999; and Hu et al., 1999).
Active caspase-9 initiates a protease cascade that also activates caspase-3.
(Fernandes-Alnemri et al., 1994) and other downstream caspases.
For a review of caspases and apoptosis, see Green & Reed, 1998 or see Cell
Death and Differentiation, Nov. 1999, Vol. 6 (11).
Figure 2 illustrates the detection of caspase activities using Caspase Assay Plates.
The different caspase substrates are composed of short peptide sequences
that are recognized by their respective activated caspases. These substrates
are covalently linked to the fluorogenic dye 7-amino-4-methyl coumarin (AMC).
Peptide-bound AMC emits in the UV range (max=380 nm). However, if the substrate
is incubated with its corresponding activated caspase, the caspase cleaves off
the fluorogenic dye (AMC). The released dye has an emission maximum of 460
nm (green). Therefore it is possible to correlate an increase in fluorescence
intensity of AMC at 460 nm with an increased activity of the respective caspase
in the tested sample.
The Caspase Assay Plates are provided with specific caspase inhibitors for each
of the caspases that can be detected. When assaying whole cell lysates, the
inhibitor can be used as a control. Inhibitors are also available separately for
investigating the overall roles of proteases in the apoptotic process. See Related
Products (Section VI) for ordering information.
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ApoAlert® Caspase Assay Plates User Manual
I. Introduction continued
Caspase3
Caspase8
Caspase9
Caspase2
A
B
C
D
E
F
G
H
Figure 1. Diagram of the Caspase Profiling Plate layout.
Induction of apoptosis in cells
Protease activation
Cell lysate preparation
Caspase-
Caspase-3
Caspase-
Caspase-9
VDVAD-AMC
DEVD-AMC
IETD-AMC
LEHD-AMC
VDVAD
IETD
DEVD
AMC
AMC
LEHD
AMC
AMC
fluorometric detection
Figure 2. Detecting protease activity using ApoAlert® Caspase Assay Plates. Fluorometric
detection of caspase activities is read with a 380-nm excitation filter and 460-nm emission filter.
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ApoAlert® Caspase Assay Plates User Manual
II. List of Components
Store all components at –20°C. Protect Caspase Inhibitors from light.
Caspase-3 Assay Plate
1 plate
5 plates
(630223)
(630224)
•
1 5 Caspase-3 Plate(s)
•
6 ml 30 ml 2X Reaction Buffer
• 100 µl 500 µl DTT (1 M)
•
6 ml 30 ml 1X Cell Lysis Buffer
• 20 µl 100 µl Caspase-3 Inhibitor (10 µM)
Caspase Profiling Assay Plate
1 plate
5 plates
(630225)
(630226)
•
1 5 Caspase Profiling Plate(s)
•
6 ml 30 ml 2X Reaction Buffer
• 100 µl 500 µl DTT (1 M)
•
6 ml 30 ml 1X Cell Lysis Buffer
• 20 µl 100 µl Caspase-3 Inhibitor (10 µM)
• 20 µl 100 µl Caspase-8 Inhibitor (10 µM)
• 20 µl 100 µl Caspase-9 Inhibitor (200 µM)
• 20 µl 100 µl Caspase-2 Inhibitor (10 µM) III. Additional Materials Required
The following materials are required but not supplied.
• Centrifuge for collecting cells
• 96-well plate reader
• Parafilm
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ApoAlert® Caspase Assay Plates User Manual
IV. Caspase Assay Plate Protocol
PLEASE READ ENTIRE PROTOCOL BEFORE BEGINNING.
A. General Considerations
A relatively high concentration of DTT is required for full activity of the
enzymes. Ensure that DTT is added to the Reaction Buffer when the assay
is performed. Otherwise, the detected caspase activity will be reduced.
Reagents:
• Make sure that all buffers are completely in solution before use.
• Protect Caspase Inhibitors from light.
• Aliquot a sufficient volume of 2X Reaction Buffer for the number of assays
you will perform (50 µl/well). Immediately before use, add DTT to the 2X
Reaction Buffer: add 10 µl of 1 M DTT stock per 1 ml 2X Reaction Buffer.
Do not add DTT to the stock 2X Reaction Buffer.
Controls:
• We recommend performing three control reactions:
1)A negative control on uninduced cells.
2)A positive control for caspase induction. You may treat your own
cells with a known inducer. At Clontech, we use either Staurosporin (700 nM) for 5 hr, or UV light.
3)[Optional] A control on induced cells treated with Caspase Inhibitor.
• Comparing the emission of an apoptotic sample with an uninduced control
allows determination of the fold-increase in protease activity.
Cells:
Use the lysate of 2 x 105 cells per well.
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Version # PR24952
ApoAlert® Caspase Assay Plates User Manual
IV. Caspase Assay Plate Protocol continued
B. Assay Procedure
1.Induce apoptosis in cells by desired method. Remember to incubate a
concurrent control culture without induction.
Set up duplicate wells for the following samples: induced, uninduced
(negative control), and induced plus inhibitor (optional).
2.Harvest and count the cells.
3.Calculate the volume of cell suspension that contains the number of
cells necessary for the number of wells that will be used for the assay
(Analyze 2 x 105 cells per well).
4.Gently pellet the cells by centrifuging at 400 x g for 5 min. Discard the
supernatant.
5.Resuspend the pellet in the correct volume of iced 1X Cell Lysis Buffer
(50 µl/2 x 105 cells). Incubate the cells on ice for 10 min.
6.Centrifuge cell lysates in a microcentrifuge at maximum speed for 5
min at 4°C to precipitate cellular debris. Transfer the supernatants to
new microcentrifuge tubes on ice.
Notes
• You may skip the centrifugation step and use the crude lysates in step 9. However, first
perform Steps 7 and 8.
• For an induced sample + Caspase Inhibitor control, transfer 50 µl cell lysate to a
separate tube and add 1 µl Caspase Inhibitor. These controls should be kept on ice
for at least 10 min before adding them to the plate wells in Step 9.
7.Add 50 µl of 2X Reaction Buffer/DTT Mix (Section IV.A) to each well of
the 96-well plate that will be used in the experiment.
8.Cover the plate with Parafilm to avoid evaporation and then preincubate the plate with the 2X Reaction Buffer/DTT Mix at 37°C for
5 min.
9.Transfer 50 µl of the appropriate cell lysate(s) from the tubes on ice to
the wells. Seal the plate with parafilm and incubate at 37°C for 2 hr in
an incubator (the use of a water bath is not recommended).
10.Analyze the plate in a fluorescent plate reader (excitation: 380 nm;
emission: 460 nm).
[Optional]: Reseal the plate and continue to incubate at 37°C in an
incubator if you wish to increase the reading. However, incubation for
more than three hours is not recommended.
11.If you have not used all of the 96 wells, after the final reading of the
Caspase Plate, you can seal the plate with Parafilm and store it at
–20°C until the remaining wells are used.
Clontech Laboratories, Inc. www.clontech.com
Protocol # PT3671-1
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ApoAlert® Caspase Assay Plates User Manual
V. References
Cain, K., Brown, D. G., Langlais, C. & Cohen, G. M. (1999) Caspase activation involves the formation of
the aposome, a large (~700 kDa) caspase-activating complex. J. Biol. Chem. 274:22686–22692.
Cohen, G. M. (1997) Caspases: the executioners of apoptosis. Biochem J. 326:1–16.
Melino, G., Ed. (1999). Cell Death & Differentiation, Volume 6 (11) Apoptosis (The Friary Press,
Dorchester, Dorset).
Fernandes-Alnemri, T., Litwack, G. & Alnemri, E. S. (1994) CPP32, a novel human apoptotic protein
with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1
beta-converting enzyme. J. Biol. Chem. 269:30761–30764.
Fraser, A. & Evan, G. (1996) A license to kill. Cell 85:781–784.
Green, D. R. & Reed, J. C. (1998) Mitochondria and apoptosis. Science 281:1309–1312.
Guo, Y., Srinivasula, S. M., Druilhe, A., Fernandes-Alnemri, T. & Alnemri, E. S. (2002) Caspase-2 induces
apoptosis by releasing proapoptotic proteins from mitochondria. J. Biol. Chem. 277(16):13430-7.
Hu, Y., Benedict, M. A., Ding, L. & Nunez, G. (1999) Role of cytochrome c and dATP/ATP hydrolysis
in Apaf-1 mediated caspase-9 activation and apoptosis. EMBO J. 18:3586–3595.
Muzio, M., Salvesen, G. S. & Dixit, V. M. (1997) FLICE induced apoptosis in a cell-free system. J.
Biol. Chem. 272:2952–2956.
Nicholson, D. W. & Thornberry, N. A. (1997) Caspases: killer proteases. Trends Biochem. Sci.
22:299–306.
Paroni, G., Henderson, C., Schnieder, C. & Brancolini, C. (2001) Caspase-2-induced apoptosis is
dependent on caspase-9, but its processing during UV- or tumor necrosis factor-dependent cell death
requires caspase-3. J. Biol. Chem. 276:21907–21915.
Porter, A. G. & Janicke, R. U. (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Diff.
6:99–104.
Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T., Litwack, G. & Alnemri, E. S. (1996). Molecular
ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease MCH-5 is a CrmA-inhibitable
protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl. Acad. Sci. USA
93:14486–14491.
Thornberry, N. A. & Littlewood, Y. (1998) Caspases: Enemies Within. Science 281:1312–1316.
Zou, H., Li, Y., Liu, X. & Wang, X. (1999) An APAF-1 cytochrome c multimeric complex is a functional
apoptosome that activates procaspase-9. J. Biol. Chem. 274:11549–11556.
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Version # PR24952
ApoAlert® Caspase Assay Plates User Manual
VI. Related Products
For the latest and most complete listing of all Clontech products, please visit
www.clontech.com.
Products
Cat. No.
ApoAlert® Apoptosis Inhibiting Reagents
•
Caspase-8 Inhibitor, IETD-fmk
630209
•
Caspase-3 Inhibitor, DEVD-CHO 630204
•
Caspase-3 Inhibitor, DEVD-fmk
630207
•
Caspase-1 Inhibitor, YVAD-cmk
630205
•
Caspase Inhibitor, VAD-fmk
630208
•
Caspase-9/6 Fluorescent Assay Kit
630211
630212
•
Caspase-8 Assay Kits
Fluorescent
630218
630219
Colorimetric
630220
630221
•
Caspase-3 Assay Kits
Fluorescent
630214 630215
Colorimetric
630216
630217
•
Mitochondrial Membrane Sensor Kit
630106
•
DNA Fragmentation Assay Kit
630107
630108
•
Annexin-V Apoptosis Kits many
•
Cell Fractionation Kit
630105
•
pDsRed2-Bid Vector
632419
ApoAlert® Apoptosis Assay Kits
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Protocol # PT3671-1
Version # PR24952
ApoAlert® Caspase Assay Plates User Manual
VI. Related Products (Cont.)
Products
Cat. No.
•
Glutathione Detection Kit 630103,
•
PARP Monoclonal Antibody (IgG1, C-2-10)
630210
•
Human TNF-α
630203
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