Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors*

Protocol summary

1. Prepare cells with test compounds
2. Add equal volume of caspase working solution
3. Incubate at room temperature for 30 min to 1 hour
4. Monitor fluorescence intensity

Important notes
Thaw kit components at room temperature before use.

1. Single caspase activity working solution
Make caspase 3/7, caspase 8 or caspase 9 working solution by adding 50 µL of substrate (Component A, B or C) into 10 mL of Assay Buffer (Component D), and mix them well.

2. Dual- or tri- caspase activity working solution:
Add 50 µL of each interested caspase substrate into 10 mL of Assay Buffer (Component D) together to make the working solution. Note: Please prepare the tested substrate solutions and the needed volume proportionally.

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/well of 10X test compounds (96-well plate) or 5 µL/well of 5X test compounds (384-plate) into PBS or the desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
   
   
2. Incubate the cell plate in a 37°C, 5% CO₂, 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 or 25 µL/well/384-well plate of desired caspase assay working solution.
   
   
4. Incubate the plate at room temperature for at least 30 to 60 min, protected from light. Note: If desired, add 1 µL of 1 mM caspase inhibitor to selected samples 10 minutes before adding the assay loading solution at room temperature to confirm the inhibition of caspase activities.
   
   
5. Monitor the fluorescence intensity as indicated in the table 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.

Table 1. Spectral wavelengths for Caspases 3/7, 8, and 9.