The detection of caspase 3/7 activity in Jurkat cells with ApoSight™ Green Caspase 3/7 substrate
. Jurkat cells (200,000 cells/well/ 96-well plate) were treated with 1 μM Staurosporine or DMSO for 4 hours. Cells were incubated with Caspase 3/7 Substrate working solution at 37°C for 1 hour. Images were taken with a fluorescence microscope using a FITC filter set.
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.
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.
|Apoptosis||Initiator||Caspase 2||Human & Mouse|
|Apoptosis||Initiator||Caspase 8||Human & Mouse|
|Apoptosis||Initiator||Caspase 9||Human & Mouse|
|Apoptosis||Effector||Caspase 3||Human & Mouse|
|Apoptosis||Effector||Caspase 6||Human & Mouse|
|Apoptosis||Effector||Caspase 6||Human & Mouse|
|Pyroptosis||Inflammatory||Caspase 1||Human & Mouse|
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
|IETD||Ile-Glu-Thr-Asp||Caspase 8, Granzyme B|
|DEVD||Asp-Glu-Val-Asp||Caspase 3, 6, 7, 8, or 10|
|VAD||Val-Ala-Asp||Caspase 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.
|Ac-DEVD-pNA *CAS 189950-66-1*||Caspase 3||405 nm||Yellow||5 mg||13405|
|Z-DEVD-pNA||Caspase 3||405 nm||Yellow||5 mg||13422|
|Z-IETD-pNA *CAS 219138-21-3*||Caspase 8, Granzyme B||405 nm||Yellow||5 mg||13413|
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.
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.
|Ac-DEVD-AFC *CAS 201608-14-2*||Caspase 3, 7||376||482||17000||0.53||5 mg||13401|
|Ac-DEVD-AMC *CAS 169332-61-0*||Caspase 3, 7||341||441||19000||N/D||5 mg||13402|
|Z-DEVD-AFC||Caspase 3, 7||376||482||17000||0.53||5 mg||13420|
|Z-DEVD-AMC *CAS 1135416-11-3*||Caspase 3, 7||341||441||19000||N/D||5 mg||13421|
|(Z-DEVD)2-R110 *CAS 223538-61-2*||Caspase 3, 7||500||522||80000||N/D||1 mg||13430|
|Z-DEVD-ProRed™ 620||Caspase 3, 7||532||619||N/D||N/D||1 mg||13433|
|Ac-IETD-AFC *CAS 211990-57-7*||Caspase 8, Granzyme B||376||482||17000||0.53||5 mg||13410|
|Ac-IETD-AMC||Caspase 8, Granzyme B||341||441||19000||N/D||5 mg||13411|
|Z-IETD-AFC *CAS 219138-02-0*||Caspase 8, Granzyme B||376||482||17000||0.53||5 mg||13425|
|(Ac-IETD)2-R110||Caspase 8, Granzyme B||500||522||80000||N/D||1 mg||13431|
- Ext. Coeff. = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
- FQY = fluorescence quantum yield in aqueous buffer (pH 7.2).
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.
|Z-VAD-FMK||Caspase 1, 2, 3, 6, 8, 9, 10||Irreversible||-||-||1 mg||13300|
|Z-DEVD-FMK||Caspase 3||Irreversible||-||-||1 mg||13303|
|Z-IETD-FMK||Caspase 8||Irreversible||-||-||1 mg||13305|
|Z-LETD-FMK||Caspase 8||Irreversible||-||-||1 mg||13307|
|Ac-DEVD-CHO *CAS 169332-60-9*||Caspase 3, 7||Reversible||-||-||1 mg||13403|
|Ac-IETD-CHO *CAS 191338-86-0*||Caspase 8||Reversible||-||-||5 mg||13412|
|mFluor™ 450-VAD-FMK||Caspase 1, 2, 3, 6, 8, 9, 10||Irreversible||406||445||25 tests||13475|
|mFluor™ 510-VAD-FMK||Caspase 1, 2, 3, 6, 8, 9, 10||Irreversible||412||505||25 tests||13476|
|FITC-C6-DEVD-FMK||Caspase 3, 7||Irreversible||491||516||1 mg||13406|
|FITC-C6-DEVD-FMK||Caspase 3, 7||Irreversible||491||516||100 µg||13408|
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 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
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*||Microplate, HCS||360||570||200 tests||22795|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence*||Microplate, HCS||490||525||200 tests||22796|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Red Fluorescence*||Microplate, HCS||540||620||200 tests||22797|
|Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*||Flow Cytometer||488||530||100 tests||22823|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue Fluorescence*||Microplate, HCS||370||450||200 tests||22812|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Green Fluorescence*||Microplate, HCS||490||525||200 tests||22798|
|Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red Fluorescence*||Microplate, HCS||540||620||100 tests||22816|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Blue Fluorescence*||Microplate, HCS||375||435||200 tests||22813|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Green Fluorescence*||Microplate, HCS||490||525||200 tests||22798|
|Cell Meter™ Caspase 9 Activity Apoptosis Assay Kit *Red Fluorescence*||Microplate, HCS||540||620||100 tests||22816|
Solution-Based Caspase 3/7 Activity Assays
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.
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, which 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)
Table 7. Solution-based assays for measuring caspase 3/7 activity.
|Amplite® Colorimetric Caspase 3/7 Assay Kit *Yellow Color*||Cell Extracts||Absorbance Microplate Reader||490||None||200 tests||13507|
|Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*||Cell Extracts||Fluorescence Microplate Reader||350||450||500 tests||13502|
|Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*||Cell Extracts||Fluorescence Microplate Reader||490||525||500 tests||13503|
|Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*||Cell Extracts||Fluorescence Microplate Reader||535||620||100 tests||13504|
Live Cell Caspase Binding Assays
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.
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 Green™ DCS1 (Necrotic Cells Stain)
- Washing Buffer
Table 8. Binding assays for measuring caspase 1, 2, 3/7, 6, 8, 9, 10, or 13 activity.
|Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*||Caspase 1||490||525||FITC||FL1||25 Tests||20108|
|Cell Meter™ Live Cell Caspase 2 Binding Assay Kit *Green Fluorescence*||Caspase 2||490||525||FITC||FL1||25 Tests||20111|
|Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*||Caspase 3/7||490||525||FITC||FL1||25 Tests||20100|
|Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*||Caspase 3/7||550||595||TRITC||FL1||25 Tests||20101|
|Cell Meter™ Live Cell Caspase 6 Binding Assay Kit *Green Fluorescence*||Caspase 6||490||525||FITC||FL1||25 Tests||20113|
|Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*||Caspase 8||490||525||FITC||FL1||25 Tests||20115|
|Cell Meter™ Live Cell Caspase 9 Binding Assay Kit *Green Fluorescence*||Caspase 9||490||525||FITC||FL1||25 Tests||20117|
|Cell Meter™ Live Cell Caspase 10 Binding Assay Kit *Green Fluorescence*||Caspase 10||490||525||FITC||FL1||25 Tests||20119|
|Cell Meter™ Live Cell Caspase 13 Binding Assay Kit *Green Fluorescence*||Caspase 13||490||525||FITC||FL1||25 Tests||20125|
|Cell Meter™ Generic Fluorimetric Caspase Binding Assay Kit *Red Fluorescence Optimized for Flow Cytometry*||Caspase-1, 3/7, 4, 5, 6, 8, 9||649||664||-||APC||25 Tests||22822|
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.
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.
|Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors*||100 Tests||22850|
Product Ordering Information
Table 10. Ordering Info for Caspase Substrates and Inhibitors Products
|13401||Ac-DEVD-AFC *CAS 201608-14-2*||5 mg|
|13402||Ac-DEVD-AMC *CAS 169332-61-0*||5 mg|
|13403||Ac-DEVD-CHO *CAS 169332-60-9*||1 mg|
|13405||Ac-DEVD-pNA *CAS 189950-66-1*||5 mg|
|13410||Ac-IETD-AFC *CAS 211990-57-7*||5 mg|
Table 11. Ordering Info for Caspase Activity and Binding Assays Products
|13502||Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*||500 Tests|
|13503||Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*||500 Tests|
|13504||Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*||100 tests|
|13507||Amplite® Colorimetric Caspase 3/7 Assay Kit *Yellow Color*||200 Tests|
|20100||Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Green Fluorescence*||25 Tests|
|20101||Cell Meter™ Live Cell Caspase 3/7 Binding Assay Kit *Red Fluorescence*||25 Tests|
|20108||Cell Meter™ Live Cell Caspase 1 Binding Assay Kit *Green Fluorescence*||25 Tests|
|20111||Cell Meter™ Live Cell Caspase 2 Binding Assay Kit *Green Fluorescence*||25 Tests|
|20113||Cell Meter™ Live Cell Caspase 6 Binding Assay Kit *Green Fluorescence*||25 Tests|
|20115||Cell Meter™ Live Cell Caspase 8 Binding Assay Kit *Green Fluorescence*||25 Tests|