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

Protocol summary

1. Prepare cells with test compounds
2. Add equal volume of caspase 3/7 working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity at Ex/Em = 535/620 nm

Important notes
Thaw Component A, B, C (if desired, Component D) at room temperature before use.

1. Z-DEVD-ProRed™ stock solution (200X):
Add 65 µL of DMSO (not provided) into the vial of Component A.

2. (Optional) Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO directly to the vial of Ac-DEVD-CHO (Component D). This inhibitor can be used to confirm the correlation between fluorescence signal intensity and caspase 3/7-like protease activities.

Add 50 μL of 200X Z-DEVD-ProRed™ stock solution and 100 μL of 1M DTT solution (Component C) into 10 mL Assay Buffer (Component B) and mix well.

Note: 50 μL of the 200X Z-DEVD-ProRed™ stock solution is enough for 100 assays using a reaction volume of 100 μL per assay.

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

1. Treat cells by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) into PBS or desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
   
   
2. Incubate the cell plates in an incubator for a desired period of time (3 - 5 hours for Jurkat cells treated with staurosporine) 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, kept from light. Note: If desired, add 1 µL of the 1 mM stock solution of the caspase 3/7 Inhibitor Ac-DEVD-CHO into selected samples 10 minutes before adding the caspase 3/7 assay working solution at room temperature to confirm the caspase 3/7-like activities.
   
   
5. Monitor the fluorescence intensity at Ex/Em = 535/620 nm (cut off at 610 nm) with either top or bottom read mode. Note: Sometimes, bottom read gives better signal to background ratio, centrifuge cell plate (especially for the nonadherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.