Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
2. Add equal volume of Caspase 3/7 working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important notes
Thaw component A, B, C (if desired, Component D and E) at room temperature before starting the experiment.

1. Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO (not provided) directly to the vial of Ac-DEVD-CHO (Component D), and mix well to make 1 mM Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution. This Caspase 3/7 Inhibitor can be used to confirm the correlation between fluorescence signal intensity and Caspase 3/7-like protease activities.

Add 50 μL of 200X Caspase 3/7 Substrate stock solution (Component A) and 100 μL of 1M DTT solution (Component C) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 3/7 working solution. Note: This Caspase 3/7 working solution is enough for 100 assays using a reaction volume of 100 μL per assay. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Treat cells with 10 µL of 10X test compound (for a 96-well plate) or 5 µL of 5X test compound (for a 384-well plate) in PBS or desired buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
   
   
2. Incubate the cell plates in an incubator for desired period of time ( 4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.
   
   
3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 working solution.
   
   
4. Incubate the plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of 1 mM Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the Caspase 3/7-like activities.  Note: If desired, prepare a R110 standards by diluting 5 mM  R110 Standard (Component E) into growth Medium to yield serially diluted R110 standards ranging from 0 - 50 µM. Add 100 µL of the serially diluted R110 standards into the wells containing 100 µL of Caspase 3/7 working solution at any time prior to measuring the fluorescence. This standard curve could be used to determine the moles of product produced in the caspase 3/7 containing reactions.
   
   
5. Centrifuge cell plates (especially for the non-adherent cells) at 800 rpm for 2 minutes with brake off. 
   
   
6. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm).