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Caspases

Caspases (cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes whose functions are intimately linked with the processes of apoptosis (programmed cell death), necrosis, and pyroptosis (inflammation). AAT Bioquest offers a plethora of reagents and kits for measuring caspase activity in real-time enzyme kinetic and end-point assay formats.

 



What Are Caspases?


Caspases are cytosolic aspartate-specific cysteine proteases that serve as the primary mediators of apoptosis. A variety of receptors, such as the TNF-α receptor, FasL receptor, TLR, and death receptors, and the Bcl-2 and inhibitor of apoptosis (IAP) protein families, participate and regulate this caspase-dependent apoptosis pathway. Once activated by an upstream stimulus (extrinsically or intrinsically), caspases execute the proteolysis of downstream protein substrates, triggering a 'cascade' of events that lead to cell disassembly, death, and phagocytosis and removal of cellular debris.

Human Caspases

The human caspase family is divided into three main groups, primarily based on commonalities such as sequence similarity and biological function. Group 1 comprises 'inflammatory' caspases with a long caspase-recruitment domain and affinity for large aromatic or hydrophobic residues at the P4 position. Group 2 comprises 'apoptotic effector' caspases with a short pro-domain. In contrast, group 3 comprises 'apoptotic initiator' caspases with a long pro-domain and affinity for substrates with leucine or valine at the P4 position (Table 1).

Initiator and Effector Caspases

Depending on their role in the apoptosis caspase pathway, caspases can be divided into two categories: initiator and effector caspases. Both initiator and effector caspases bear a catalytic site comprised of a small and a large subunit. The recognition site for caspases is marked by three to four amino acids followed by an aspartic acid residue, with the cleavage occurring after the aspartate. The caspase proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspases through cleavage at internal aspartates, either by autocatalysis or by the action of another protease. Once activated, initiator caspases cleave and activate effector caspases. Activated effector caspases subsequently cleave several protein substrates to trigger apoptosis.

Apoptotic initiator caspases such as caspase-2, -8, -9, and -10 can initiate the caspase activation cascade. Caspase-8 is essential for the formation of the death-inducing signaling complex (DISC). When activated, caspase-8 activates downstream effector caspases (e.g., caspase 3) and mediates the release of cytochrome c from the mitochondria. Caspase-8 has proven to show relatively high substrate selectivity to the IETD peptide sequence. Apoptotic effector caspases such as caspase-3, -6, and -7 may not be responsible for initiating the cascading pathway. Still, when activated, they play an integral role in the intermediate and later steps of the cascade. Caspase-3 (CPP32/apopain) is a key effector as it amplifies the signal from an initiator caspases and signifies the full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase 3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase Cδ and actin. Detection of activated caspase-3 is carried out using a DEVD peptide sequence which is selective for caspase-3 and proven successful at developing caspase-3 substrates.

 

Table 1. Functional classification of human caspases.

Cell Death Pathway
Type of Caspase
Enzyme
Organism
ApoptosisInitiatorCaspase 2Human & Mouse
ApoptosisInitiatorCaspase 8Human & Mouse
ApoptosisInitiatorCaspase 9Human & Mouse
ApoptosisInitiatorCaspase 10Human
ApoptosisEffectorCaspase 3Human & Mouse
ApoptosisEffectorCaspase 6Human & Mouse
PyroptosisInflammatoryCaspase 1Human & Mouse
PyroptosisInflammatoryCaspase 4Human
PyroptosisInflammatoryCaspase 5Human
PyroptosisInflammatoryCaspase 11Mouse



Caspase Substrates & Inhibitors


Caspase substrates and inhibitors consist of two key components: a caspase recognition sequence and either a signal generating or a protease inhibiting motif. The caspase recognition sequence is marked by three or four amino acids specific to the caspase or caspases being measured (Table 2). The N-terminus of a caspase recognition sequence are typically modified with either acetyl (Ac) or carbobenzoxy (Z) groups to enhance membrane permeability. The intended caspase recognizes the specific peptide sequence as its enzymatic cleavage site, releasing the signal generating or inhibiting motif. Both chromogenic and fluorogenic caspase substrates operate in a similar fashion in which signal or color intensity is proportional to the proteolytic activity.

 

Table 2. Peptide recognition sequences for caspase activity For Caspase Substrates & Inhibitors

Peptide
Amino Acid Sequence
Target for Caspases
IETDIle-Glu-Thr-AspCaspase 8, Granzyme B
DEVDAsp-Glu-Val-AspCaspase 3, 6, 7, 8, or 10
LEHDLeu-Glu-His-AspCaspase 9
VADVal-Ala-AspCaspase 1, 2, 3, 6, 8, 9 or 10

 

Chromogenic Caspase Substrates

Chromogenic caspase substrates comprise a signal generating motif, such as the highly colored chromophore pNA (para-nitroaniline or 4-nitroaniline) linked to a caspase recognition sequence which is monitored colorimetrically at 405 nm using either an absorbance microplate reader or a spectrophotometer.

 

Table 3. Chromogenic caspase substrates.

Substrate
Target Caspase(s)
Abs (nm)
Color
Unit Size
Cat No.
Ac-DEVD-pNA *CAS 189950-66-1*Caspase 3405 nmYellow5 mg13405
Z-DEVD-pNACaspase 3405 nmYellow5 mg13422
Z-IETD-pNA *CAS 219138-21-3*Caspase 8, Granzyme B405 nmYellow5 mg13413

 

Fluorogenic Caspase Substrates

The structure of fluorogenic caspase substrates comprises of a fluorophore such as 7-Amino-4-methylcoumarin (AMC), 7-Amino-4-trifluoromethylcoumarin (AFC), Rhodamine 110 (R110), or ProRed™ 620 that is linked to a caspase recognition sequence. R110-based caspase substrates are more sensitive than coumarin-based caspase substrates (e.g., AMC and AFC) but have narrower dynamic ranges due to the two-step cleavage process. It is recommended that R110-based caspase substrates be used for endpoint assays while AMC and AFC caspase substrates are used for kinetic assays.

 

AFC, AMC, R110 and ProRed™ 620 spectra
Figure 1. From left to right, the excitation and emission spectra of AMC (7-Amino-4-methylcoumarin), AFC (7-Amino-4-trifluoromethylcoumarin), Rhodamine 110(R110), and ProRed™ 620.

 

Table 4. Fluorogenic caspase substrates.

Substrate
Target for Caspases
Ex (nm)
Em (nm)
ε¹
Φ²
Unit Size
Cat No.
Ac-DEVD-AFC *CAS 201608-14-2*Caspase 3, 7376482170000.535 mg13401
Ac-DEVD-AMC *CAS 169332-61-0*Caspase 3, 734144119000N/D5 mg13402
Z-DEVD-AFCCaspase 3, 7376482170000.535 mg13420
Z-DEVD-AMC *CAS 1135416-11-3*Caspase 3, 734144119000N/D5 mg13421
(Z-DEVD)2-R110 *CAS 223538-61-2*Caspase 3, 750052280000N/D1 mg13430
Z-DEVD-ProRed™ 620Caspase 3, 7532619N/DN/D1 mg13433
Ac-IETD-AFC *CAS 211990-57-7*Caspase 8, Granzyme B376482170000.535 mg13410
Ac-IETD-AMCCaspase 8, Granzyme B34144119000N/D5 mg13411
Z-IETD-AFC *CAS 219138-02-0*Caspase 8, Granzyme B376482170000.535 mg13425
(Ac-IETD)2-R110Caspase 8, Granzyme B50052280000N/D1 mg13431
  1. Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).

 

Caspase Inhibitors

Caspase inhibitors bind to the active site of caspases and form either a reversible or irreversible linkage. Generally, caspase inhibitors' structure consists of the caspase recognition sequence and a functional group such as aldehyde (-CHO) or fluoromethylketone (-FMK). Caspase inhibitors with an aldehyde functional group are reversible, whereas inhibitors which have FMK are irreversible. Both caspase substrates and inhibitors have minimal cytotoxic effects and, therefore, are useful tools when studying caspase activity.

 

Table 5. Reversible and irreversible binding caspase inhibitors.

Inhibitor
Target for Caspases
Binding
Ex (nm)
Em (nm)
Unit Size
Cat No.
Ac-DEVD-CHO *CAS 169332-60-9*Caspase 3, 7Reversible--1 mg13403
Ac-IETD-CHO *CAS 191338-86-0*Caspase 8Reversible--5 mg13412
mFluor™ 450-VAD-FMKCaspase 1, 2, 3, 6, 8, 9, 10Irreversible40644525 tests13475
mFluor™ 510-VAD-FMKCaspase 1, 2, 3, 6, 8, 9, 10Irreversible41250525 tests13476
FITC-C6-DEVD-FMKCaspase 3, 7Irreversible4915161 mg13406
FITC-C6-DEVD-FMKCaspase 3, 7Irreversible491516100 µg13408
FITC-C6-LEHD-FMKCaspase 9Irreversible4915161 mg13407
FITC-C6-LEHD-FMKCaspase 9Irreversible491516100 µg13409
FAM-VAD-FMKCaspase 1, 2, 3, 6, 8, 9, 10Irreversible49351725 tests13470
SRB-VAD-FMK [Sulforhodamine B-VAD-FMK]Caspase 1, 2, 3, 6, 8, 9, 10Irreversible55957725 tests13472



Caspase 3, 7, 8 and 9 Activity Assays


Cell Meter™ Caspase Activity Assay kits provide a robust and convenient method for measuring caspase activity in cells. These assays use substrates that contain recognition sites specific to measuring caspases 3 and 7, caspase 8, or caspase 9 activation, as well as a multiplex assay designed to measure all four caspases simultaneously. Conjugation of the caspase recognition sequence inhibits the fluorophore or chromophore's ability to produce a quantifiable signal. In the presence of an appropriately activated caspase, the fluorophore or chromophore is cleaved from the recognition sequence and free to generate either a bright fluorescent or colored signal indicative of apoptosis. Cell Meter™ Caspase Activity Assays are robust and highly specific for their respective activated caspase and can be readily adapted for high throughput screening. Using 100 µL of reagents per well in a 96-well format, this kit provides sufficient reagents to perform 200 assays. Using 25 µL of reagents per well in a 384-well format, this kit provides sufficient reagents to perform 800 assays.


Detection of caspase 3, 7, 8 and 9 activities
Figure 2. Detection of Caspase Activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 mM for 4 hours (Red Bar), while the untreated cells were used as control (Blue Bar). The single-caspase assay loading solution (100 µL/well) was added (in #1 for caspase 3/7, #2 for caspase 8 or #3 for caspase 9), or Triple-caspase assay loading solution (#4 for caspase 3/7, 8, and 9 together) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured with a FlexStation fluorescence microplate reader at the indicated wavelength. The caspase 3/7, 8, and 9 activities can be detected in a single assay without interferences from other caspases.

 

Table 6. Assay kits for measuring Caspase 3, 7, 8 and 9 activity

Kit
Platform
Ex (nm)
Em (nm)
Unit Size
Cat No.
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS360570200 tests22795
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22796
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620200 tests22797
Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*Flow Cytometer488530100 tests22823
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS370450200 tests22812
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22798
Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620100 tests22816
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*Microplate, HCS375435200 tests22813
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*Microplate, HCS490525200 tests22798
Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence*Microplate, HCS540620100 tests22816



Solution-Based Caspase 3/7 Activity Assays


The Amplite™ Caspase 3/7 Activity Assay Kit provides a simple and robust method to measure caspase 3 and 7 in cell extracts and purified enzyme preparations. The assays use substrate containing a DEVD recognition sequence specific for caspase 3/7—conjugation of the DEVD recognition sequence masks the signal output of the attached fluorophore or chromophore. In the presence of caspase 3/7, the fluorophore or chromophore is cleaved from the DEVD sequence and free to produce either a bright fluorescence or colored signal indicative of apoptosis.

Fluorimetric caspase 3/7 activity assay kits included:

  • Caspase 3/7 Substrate (200X Stock Solution)
  • Caspase 3/7 Inhibitor
  • Assay Buffer
  • DTT (Reducing agent)

 

Caspase 3/7 Activity
Figure 3. Detection of Caspase 3/7 activity in Jurkat cells with Amplite™ Fluorimetric Caspase 3/7 Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 20 µM of camptothecin for 5 hours and with or without 5 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm.

 

Table 7. Solution-based assays for measuring caspase 3/7 activity.

Product Name
Sample Type
Platform
Ex (nm)
Em (nm)
Unit Size
Cat No.
Amplite™ Colorimetric Caspase 3/7 Assay Kit *Yellow Color*Cell ExtractsAbsorbance Microplate Reader490None200 tests13507
Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*Cell ExtractsFluorescence Microplate Reader350450500 tests13502
Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*Cell ExtractsFluorescence Microplate Reader490525500 tests13503
Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*Cell ExtractsFluorescence Microplate Reader535620100 tests13504



Live Cell Caspase Binding Assays


In apoptosis, one of the key events is the activation of caspases, which is important for the initiation of apoptosis. Cell Meter™ Live Cell Caspase Binding Kits use fluorescent cell-permeable and nontoxic indicators to detect caspases 1, 2, 3/7, 6, 8, 9, 10, and 13 activities. Once bound to caspases, the fluorescent reagents are retained inside the cell. The binding event prevents the caspases from further catalysis but will not stop apoptosis from proceeding. The caspase binding kits are applicable for fluorescence microscope, flow cytometer, and fluorescence microplate reader. The kits provide all the essential components with an optimized assay protocol, as well as a Hoechst dye (Ex/Em = 350/461 nm) for labeling the whole population of cells and a propidium iodide dye (Ex/Em = 535/635) or Nuclear Green™ DCS1 (Assay Kit 20101) for staining necrotic cells.

Caspase binding assays kits included:

  • Fluorescent Caspase Inhibitor
  • 500X Hoechst (Live Cell DNA Stain)
  • 500X Propidium Iodide or 500X Nuclear Grean™ DCS1 (Necrotic Cells Stain)
  • Washing Buffer

 

Flow Caspase 3/7 Activity
Figure 4. Fluorometric detection of active caspases 3/7 using FAM-DEVD-FMK (Cat No. 20100) in Jurkat cells. The cells were treated with 1 µM staurosporine for 4 hours (Red), while untreated cells were used as a control (Green). Control and treated cells were incubated with FAM-DEVD-FMK for 1 hour at 37 °C and then washed once after stain. Fluorescent intensity was measured with NovoCyte™ 3000 Flow Cytometer FITC channel.

 

Table 8. Binding assays for measuring caspase 1, 2, 3/7, 6, 8, 9, 10, or 13 activity.

Product Name
Target Caspase
Ex (nm)
Em (nm)
Filter Set
Channel
Unit Size
Cat No.
Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*Caspase 1490525FITCFL125 Tests20108
Cell Meter™ Live Cell Caspase 2 Binding Assay Kit *Green Fluorescence*Caspase 2490525FITCFL125 Tests20111
Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*Caspase 3/7490525FITCFL125 Tests20100
Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*Caspase 3/7550595TRITCFL125 Tests20101
Cell Meter™ Live Cell Caspase 6 Binding Assay Kit *Green Fluorescence*Caspase 6490525FITCFL125 Tests20113
Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*Caspase 8490525FITCFL125 Tests20115
Cell Meter™ Live Cell Caspase 9 Binding Assay Kit *Green Fluorescence*Caspase 9490525FITCFL125 Tests20117
Cell Meter™ Live Cell Caspase 10 Binding Assay Kit *Green Fluorescence*Caspase 10490525FITCFL125 Tests20119
Cell Meter™ Live Cell Caspase 13 Binding Assay Kit *Green Fluorescence*Caspase 13490525FITCFL125 Tests20125
Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit *Red Fluorescence Optimized for Flow Cytometry*Caspase-1, 3/7, 4, 5, 6, 8, 9649664-APC25 Tests22822

 

Caspase & Phosphatidylserine Multiplexing Assay


Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit is designed to detect apoptosis by simultaneously monitoring Caspase 3/7 and annexin V activities in mammalian cells. Annexins are a family of proteins that bind to phospholipid membranes in the presence of calcium. Annexin V is used to detect apoptotic cells that express phosphatidylserine (PS) on the cell surface. The appearance of PS on the cell surface is a universal indicator of the initial/intermediate cell apoptosis stages. Annexin V-dye conjugates monitor cell apoptosis by measuring the translocation of PS. The kit also provides a Hoechst dye for labeling the nucleus of the whole population of the cells and propidium iodide dye for staining necrosis cells.

 

Detection of caspase 3, 7, 8 and 9 activities
Figure 5. The fluorescence image analysis indicated increased caspase expression 3/7 (red, stained by TF3-DEVD-FMK) and annexin V (Green, stained by Annexin V-iFluor 488™) in Jurkat cells induced by 1 µM staurosporine for 3 hours. Fluorescence images of the cells (300,000 cells/well) were taken with an Olympus fluorescence microscope through the DAPI, FITC, and TRITC channels. Individual images taken from each channel from the same cell population were merged as shown above. A: Non-induced control cells; B: Double staining of staurosporine-induced cells for caspase 3/7 (red) and Annexin V (green); C: Triple staining of staurosporine-induced cells for caspase 3/7 (red), annexin V (green), and nuclear (blue).

 

Table 9. Caspase and phosphatidylserine detection kit for measuring apoptosis.

Product Name
Unit Size
Cat No.
Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors*100 Tests22850



Product Ordering Information


 

Table 10. Ordering Info for Caspase Substrates and Inhibitors Products

Cat#
Product Name
Unit Size
Price
13401Ac-DEVD-AFC *CAS 201608-14-2*5 mg100
13402Ac-DEVD-AMC *CAS 169332-61-0*5 mg100
13403Ac-DEVD-CHO *CAS 169332-60-9*1 mg100
13405Ac-DEVD-pNA *CAS 189950-66-1*5 mg100
13406FITC-C6-DEVD-FMK1 mg520
13407FITC-C6-LEHD-FMK1 mg520
13408FITC-C6-DEVD-FMK100 ug205
13409FITC-C6-LEHD-FMK100 ug205
13410Ac-IETD-AFC *CAS 211990-57-7*5 mg205
13411Ac-IETD-AMC5 mg205



Table 11. Ordering Info for Caspase Activity and Binding Assays Products

Cat#
Product Name
Unit Size
Price
13502Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*500 Tests310
13503Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*500 Tests310
13504Amplite™ Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*100 tests363
13507Amplite™ Colorimetric Caspase 3/7 Assay Kit *Yellow Color*200 Tests205
20100Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*25 Tests310
20101Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*25 Tests310
20108Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*25 Tests310
20111Cell Meter™ Live Cell Caspase 2 Binding Assay Kit *Green Fluorescence*25 Tests310
20113Cell Meter™ Live Cell Caspase 6 Binding Assay Kit *Green Fluorescence*25 Tests310
20115Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*25 Tests310