Microplate Assays for Caspase Activity

A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes, which participate in the cleavage of protein substrates and in the subsequent disassembly of the cell. We offer a series of caspase assays that allow the simple detection of active caspases in living cells in real time or in cellular lysates or extracts.

Live-cell assays

CellEvent® Caspase-3/7 Green Detection Reagent is optimized for apoptosis analysis with a simple no-wash protocol that helps preserve delicate apoptotic cells. Live cells in microplate wells can be monitored in real time for apoptotic activity by measuring fluorescence intensity.

Caspase-3/7 assays

Microplate assay kits for caspase-3/7 measurement provide a choice of substrates with different wavelength emissions. Kits are available using rhodamine 110 or AMC to report caspase activity. Substrates are also available as bulk reagents or predispensed into microplate wells

Other caspase substrates

Rhodamine 110–derived caspase substrates are available with a variety of specificities for different caspases. They are cleaved by the protease and converted first to the fluorescent monoamide and then to R110 with a further increase in fluorescence. The substrates can be used to continuously measure enzyme activity in cell extracts and purified enzyme preparations using a fluorescence microplate reader.

CellEvent™ Caspase-3/7 Green Detection Reagent
Dose–response curve with CellEvent™ Caspase-3/7 Green Detection Reagent. The percent positive for active caspase3/7 and EC50 were determined for staurosporine-treated U2OS cells using the reagents in the CellEvent™ Caspase-3/7 Green Detection Reagent.

Selection guides
  CellEvent® Caspase-3/7 Green Detection Reagent
Target Caspase 3/7
Reporter Proprietary dye conjugated DEVD peptide
Ex/Em (nm) 502/530
Live cell Yes
Lysate Yes
Enzyme prep No
Components Live-cell caspase 3/7 enzymatic substrate
Format 100 µL stock solution for ~400 assays
Protocol outline
  1. Dilute reagent to desired concentration.
  2. Apply to cells.
  3. Incubate 30 minutes.
  4. Measure resulting fluorescence.
Cat. No. C10423
  EnzChek® Caspase-3 Assay Kit #1, Z-DEVD-AMC Substrate EnzChek® Caspase-3 Assay Kit #2, Z-DEVD-R110 Substrate Rhodamine 110, bis-(N-CBZ-L-Aspartyl-L-Glutamyl-L-Valyl-L-Aspartic Acid Amide) (Z-DEVD-R110) RediPlate™ 96 EnzChek® Caspase-3 Assay Kit, Z-DEVD-R110 Substrate
Target Caspase-3/7 Caspase-3/7 Caspase-3/7 Caspase-3/7
Reporter Z-DEVD-AMC substrate Z-DEVD-R110 substrate Z-DEVD-R110 substrate Z-DEVD-R110 substrate
Ex/Em (nm) 342/441 496/520 496/520 496/520
Live cell No No No No
Lysate Yes Yes Yes Yes
Enzyme prep Yes Yes Yes Yes
Components Includes reference standard and inhibitor Includes reference standard and inhibitor Enzyme substrate for cell lysates Includes buffers and inhibitor
Format 500 assays using 100 µL reaction volume 500 assays using 100 µL reaction volume 20 mg 96 assays, pre-dispensed into microplate wells
Protocol outline
  1. Lyse cells, isolate supernatant.
  2. Prepare DEVD substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase activity using standard curve.
  1. Lyse cells, isolate supernatant
  2. Prepare DEVD substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase activity using standard curve.
  1. Purify samples containing caspase-3/7 activity.
  2. Prepare DEVD substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase activity using standard curve.
  1. Optimize reaction buffer.
  2. Rehydrate substrate and reference standards.
  3. Add samples to wells.
  4. Incubate.
  5. Measure fluorescence.
  6. Determine caspase activity using standard curve.
Cat. No. E13183 E13184 R22120 R35100
  Rhodamine 110, bis-(N-CBZ-L-Tyrosinyl-L-Valyl-L-Alanyl-L-Aspartic Acid Amide) (Z-YVAD-R110) Rhodamine 110, bis-(N-CBZ-L-Valyl-L-Glutamyl-L-Isoleucyl-L-Aspartic Acid Amide) (Z-VEID-R110) Rhodamine 110, bis-(N-CBZ-L-Valyl-L-Aspartyl-L-Valyl-L-Alanyl-L-Aspartic Acid Amide) (Z-VDVAD-R110)
Target Caspase 1/4 Caspase 6 Caspase 2
Reporter Z-YVAD-R110 Z-VEID-R110 Z-VDVAD-R110
Ex/Em (nm) 496/520 496/520 496/520
Live cell No No No
Lysate Yes Yes Yes
Enzyme prep Yes Yes Yes
Components Bulk reagent Bulk reagent Bulk reagent
Format 2 mg 2 mg 2 mg
Protocol outline
  1. Lyse cells.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase 1 or 4 activity using standard curve.
  1. Lyse cells.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase 6 activity using standard curve.
  1. Lyse cells.
  2. Prepare substrate and apply to wells.
  3. Add samples to wells.
  4. Incubate samples.
  5. Measure fluorescence.
  6. Determine caspase 2 activity using standard curve.
Cat. No. R33750 R33754 R33755
For Research Use Only. Not for use in diagnostic procedures.