Description
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.
