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Caspases
AAT Bioquest offers a comprehensive portfolio of caspase detection reagents for apoptosis research, including fluorogenic and luminogenic substrates, irreversible inhibitors, and complete assay kits. These tools enable detection of initiator and executioner caspase activities involved in apoptosis (including caspase-2, -3/7, -6, -8, -9, and -10), as well as selected inflammatory or stress-associated caspases such as caspase-1 and caspase-12, using multiple detection platforms. Our Cell Meter™ and Amplite® assay kits provide optimized, ready-to-use solutions with superior sensitivity and convenience.
Cell Meter™ Caspase Activity Assay Kits
Cell Meter™ Caspase Activity Assay Kits provide optimized solutions for detecting caspase activity in cell lysates using fluorogenic substrates. Upon cleavage by specific caspases, these substrates release highly fluorescent reporter molecules (AMC, R110, or ProRed™) that can be quantified using a microplate reader. Multiple fluorescence colors allow parallel measurement of selected caspase activities when compatible substrates and assay conditions are appropriately optimized.
Key Features
- Optimized buffers for maximum enzyme activity and signal
- Available in blue (AMC), green (R110), and red (ProRed™) fluorescence
- Compatible with standard fluorescence microplate readers
- Complete kits include substrates, lysis buffer, and positive controls
- Multiplexing capability with spectrally distinct substrates
Fig. 1
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.
Amplite® Caspase 3/7 Activity Assay Kits
Amplite® Caspase Assay Kits provide robust, high-throughput solutions for measuring caspase-3/7 activity. These kits utilize optimized fluorogenic or colorimetric substrates with enhanced signal-to-background ratios, making them ideal for drug screening and apoptosis research applications.
Key Features
- Superior signal-to-background for improved sensitivity
- Homogeneous "mix-and-read" format
- Available in fluorimetric (blue, green, red) and colorimetric formats
- Optimized for 96-well and 384-well microplate formats
- Stable reagents with long shelf life
Fig. 2
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.
Cell Meter™ Live Cell Caspase Binding Assay Kits
Cell Meter™ Live Cell Caspase Binding Assay Kits utilize fluorescent-labeled FMK (fluoromethyl ketone) inhibitors that irreversibly bind to active caspases in living cells. These cell-permeable probes allow real-time detection of caspase activation without cell lysis, making them ideal for flow cytometry and fluorescence microscopy applications.
Key Features
- Cell-permeable probes for live cell analysis
- Irreversible binding ensures stable signal
- No cell lysis required—preserves cell integrity
- Compatible with flow cytometry and fluorescence microscopy
- Available for specific caspases (1, 2, 3/7, 6, 8, 9, 10, 13) and pan-caspase detection
Fig. 3
Fluorometric detection of active caspases 3/7 using FAM-DEVD-FMK (Catalog Number 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.
Caspase Substrates
Fluorogenic Substrates
Fluorogenic caspase substrates consist of short peptide sequences linked to fluorescent reporter molecules (AMC, AFC, R110, or ProRed™). Upon cleavage by specific caspases, the fluorophore is released from the peptide, generating a measurable fluorescence signal proportional to enzyme activity. These substrates are available with different N-terminal blocking groups (Ac- or Z-) that affect cell permeability and enzyme kinetics.
Key Features
- AMC substrates: Blue fluorescence (Ex/Em = 341/441 nm), widely used standard
- AFC substrates: Cyan fluorescence (Ex/Em = 376/482 nm), higher quantum yield than AMC
- R110 substrates: Green fluorescence (Ex/Em = 500/522 nm), superior sensitivity
- ProRed™ substrates: Red fluorescence (Ex/Em = 532/619 nm), ideal for multiplexing
Luminogenic Substrates
Luminogenic aminoluciferin substrates provide ultra-sensitive bioluminescent detection of caspase activity. Upon caspase cleavage, aminoluciferin is released and can be detected using luciferase in a coupled reaction. These substrates offer the highest sensitivity for detecting low levels of caspase activity.
Key Features
- Ultra-high sensitivity for detection of low caspase activity
- Wide dynamic range
- Compatible with standard luminometers
- Available for multiple caspase targets
Specialty Substrates
Beyond standard caspase recognition sequences, AAT Bioquest offers specialty substrates for related proteases and unique applications. These include FRET-based substrates with MCA/Dnp pairs for continuous kinetic monitoring of selected caspases, as well as specialized substrates targeting caspase-1/ICE for inflammasome-related studies. These reagents support advanced research applications requiring real-time enzyme activity measurement or detection of non-canonical caspase activities.
Caspase Inhibitors
Caspase inhibitors are essential tools for studying apoptotic pathways and validating caspase-dependent processes. AAT Bioquest offers both irreversible (FMK) and reversible (CHO) inhibitors. FMK inhibitors form covalent bonds with the active site cysteine, while CHO (aldehyde) inhibitors provide reversible competitive inhibition. Fluorescent-labeled FMK inhibitors also serve as probes for detecting active caspases in live cells.
Key Features
- FMK inhibitors: Cell-permeable, irreversible, available unlabeled or fluorescent-labeled
- CHO inhibitors: Reversible, useful for mechanistic studies
- Pan-caspase (VAD) and specific sequences available
- Non-cytotoxic during typical experimental timeframes
Fig. 4
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).
This document (01.0092.211015r2) was last updated on Sat Feb 28 2026. All trademarks and registered trademarks mentioned herein are the property of their respective owners.