Caspase-Glo® 3/7 Assay System
Caspase Activity Assay for Apoptosis Detection
- High sensitivity caspase assay—requires fewer cells and less enzyme
- Simple add-mix-measure protocol—no sample preparation needed
- Easily scalable to 96-, 384- and 1,536-well plate formats
- Integrates seamlessly with the MyGlo® Reagent Reader
Catalog Number:
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Catalog Number: G8090
Catalog Number: G8091
Catalog Number: G8093
Catalog Number: G8092
What Is the Caspase-Glo® 3/7 Assay?
The Caspase-Glo® 3/7 Assay is a homogeneous, bioluminescent assay that measures the activity of caspase-3 and caspase-7, two key executioner caspases activated during apoptosis. It provides a quantitative, plate-based readout of caspase activity in cultured cells or purified enzyme preparations, making it a reliable tool for detecting and measuring programmed cell death.
The assay uses a proluminescent substrate containing the DEVD tetrapeptide sequence recognized by caspase-3 and caspase-7. When these caspases cleave the substrate, free aminoluciferin is released and consumed by a proprietary thermostable luciferase, generating a stable “glow-type” luminescent signal proportional to caspase activity. This single-reagent, add-mix-measure format eliminates wash steps and sample preparation, reducing hands-on time and well-to-well variability.
Using the MyGlo® Reagent Reader in conjunction with the ProNect™ Data Platform, you can visualize caspase activity with ease.
Need to measure caspase activity in 3D cultures? See our new Caspase-Glo® 3/7 3D Assay!
How Does the Caspase-Glo® 3/7 Assay Work?
The assay works in three straightforward steps.
- Add an equal volume of the single Caspase-Glo® 3/7 Reagent directly to cells in a multiwell plate. The reagent simultaneously lyses the cells and provides the luminogenic caspase substrate.
- Mix briefly and incubate at room temperature for 30 minutes to 3 hours.
- Measure luminescence on a plate reader. No cell lysis buffer, washing, or substrate addition steps are required.
The proprietary reagent formulation combines a thermostable luciferase, the DEVD-aminoluciferin substrate, and an optimized buffer system in a single ready-to-use solution. This design produces a stable luminescent signal with a half-life greater than 5 hours, giving you a wide measurement window and reducing the need to read plates immediately.
Luminescence Is Linear Over a Broad Range of Cell Numbers
Jurkat cells were treated with anti-Fas mAb for 4.5 hours to induce apoptosis or were left untreated. Caspase-Glo® 3/7 Reagent was added directly to the cells in 96-well plates and incubated for 1 hour before recording luminescence. Each point represents the average of 4 wells. The "no cell" blank control value has been subtracted from each.
Measure Dose Response to Apoptosis Inducers
Jurkat cells were seeded at 10,000 cells/well and treated for 20 hours with two compounds. Caspase-Glo® 3/7 Reagent was added to the cells and luminescence measured on the MyGlo® Reagent Reader. The dose-response graph shows an apoptotic response to bortezomib with no apoptotic response to palbociclib.
Multiplex Caspase 3/7 with Cytotoxicity and Cell Viability Assays
The Caspase-Glo® 3/7 Assay chemistry is used in the ApoTox-Glo™ Triplex Assay, which measures cell viability, cytotoxicity, and apoptosis from the same well in a single experiment. This multiplexed approach lets you assess multiple endpoints without splitting your sample, preserving precious cells and reducing plate-to-plate variability.
In the triplex workflow, you first add a fluorescent viability/cytotoxicity reagent, measure, then add the Caspase-Glo® 3/7 Reagent and measure luminescence. The sequential format avoids signal interference between fluorescent and luminescent readouts. The result is a comprehensive cell health profile that reveals whether your treatment is affecting viability, membrane integrity, or caspase-dependent apoptosis.
Notes:
Using 100μl of Caspase-Glo® Reagent per assay in a 96-well format, Cat.# G8090 provides sufficient reagents to perform 25 caspase activity assays; Cat.# G8091, 100 assays; Cat.# G8092 and G8093, 1,000 assays. Using 25μl of Caspase-Glo® Reagent per assay in a 384-well format, Cat.# G8090 provides sufficient reagents to perform 100 caspase activity assays; Cat.# G8091, 400 assays; Cat.# G8092 and G8093, 4,000 assays.
Pair with the MyGlo® Reagent Reader
Reading your assay results doesn't require a shared plate reader. The MyGlo® Reagent Reader is a compact 96-well luminescence plate reader, enabling you to run luminescent assays at your bench without competing for instrument time. Integrated software generates dose response curves, single concentration and linearity analysis in minutes.
Protocols
Complete Protocol
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
Caspase-Glo® 3/7 Buffer |
G810C | 1 × 2.5ml |
|
Caspase-Glo® 3/7 Substrate |
G811C | 1 × 1 bottle |
SDS
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
Caspase-Glo® 3/7 Buffer |
G810A | 1 × 10ml |
|
Caspase-Glo® 3/7 Substrate |
G811A | 1 × 1 bottle |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
Caspase-Glo® 3/7 Buffer |
G810A | 10 × 10ml |
|
Caspase-Glo® 3/7 Substrate |
G811A | 10 × 1 bottle |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
What's in the box?
| Item | Part # | Size |
|---|---|---|
|
Caspase-Glo® 3/7 Buffer |
G810B | 1 × 100ml |
|
Caspase-Glo® 3/7 Substrate |
G811B | 1 × 1 bottle |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
Resources
Articles
- Using a Real Time Kinetic Cytotoxicity Assay to Determine when to Detect Apoptosis by Caspase-3/7 Activation
- Profiling Compound Effects on Cell Health Using a Multiplexed Assay
- Elevated expression of miR-494-3p is associated with resistance to osimertinib in EGFR T790M-positive non-small cell lung cancer
- Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist
- Targeted zinc-finger repressors to the oncogenic HBZ gene inhibit adult T-cell leukemia (ATL) proliferation
- Metformin attenuates rotenone-induced oxidative stress and mitochondrial damage via the AKT/Nrf2 pathway
- Endogenous retroviruses mediate transcriptional rewiring in response to oncogenic signaling in colorectal cancer
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Product Citations
Recent publications that mention the use of this product.
Frequently Asked Questions
How many cells do I need per well for the Caspase-Glo® 3/7 Assay?
Cell number depends on your cell type and the expected level of caspase activation. For most adherent and suspension cell lines, 1,000 to 20,000 cells per well in a 96-well plate provides a reliable signal. Because luminescent detection has very low background, you can work with fewer cells than fluorescent assays typically require. Run a cell titration during assay optimization to determine the optimal seeding density for your system.
How long is the luminescent signal stable?
The glow-type luminescent signal has a half-life greater than 5 hours under standard conditions. This extended stability gives you a wide measurement window and means you do not need to read plates immediately after the incubation period. For best results, read plates within 3 hours of adding reagent.
Can I use the Caspase-Glo® 3/7 Assay with 3D cell culture models?
For standard 2D cultures, the Caspase-Glo® 3/7 Assay works well as described. For 3D models such as spheroids, organoids, or microtissues, we recommend the Caspase-Glo® 3/7 3D Assay, which includes enhanced lytic capacity to penetrate dense 3D structures and ensure complete cell lysis.
Does serum in the culture medium affect results?
Fetal bovine serum and other sera can contain low levels of endogenous caspase-3/7 activity, which may contribute to background signal. Always include a “no-cell” control containing medium plus serum (without cells) and subtract this background from your experimental readings. This correction ensures your results reflect caspase activity from your treated cells only.
What plate reader should I use?
Any luminometer or multimode plate reader capable of reading luminescence is compatible. We recommend the GloMax® Discover or GloMax® Navigator plate readers, which are preloaded with optimized protocols for Promega luminescent assays and do not require gain adjustments. For a streamlined benchtop option, the MyGlo® Reagent Reader pairs with the ProNect™ Data Platform for simplified data capture and visualization.
Can I multiplex the Caspase-Glo® 3/7 Assay with other assays in the same well?
Yes. The Caspase-Glo® 3/7 Assay chemistry is the apoptosis component of the ApoTox-Glo™ Triplex Assay, which sequentially measures viability (GF-AFC substrate), cytotoxicity (bis-AAF-R110 substrate), and apoptosis (Caspase-Glo® 3/7) from one well. You can also pair the Caspase-Glo® 3/7 Assay with the RealTime-Glo™ MT Cell Viability Assay or CellTox™ Green Cytotoxicity Assay for custom multiplex experiments.
