Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Blue 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 8 Substrate working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
3. Incubate at room temperature for 30 - 60 minutes
4. Monitor fluorescence increase at Ex/Em = 370/450 nm (Cutoff = 420 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

Add 50 µL of Caspase 8 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspase 8 Substrate working solution is not stable, use it promptly. Note: Aliquot and store unused Caspase 8 Substrate (Component A) and Assay Buffer (Component B) at -20 ^oC. Avoid repeated freeze/thaw cycles.

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-well 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 5% CO₂, 37°C incubator for a desired period of time (4 - 6 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 8 Substrate working solution.
   
   
4. Incubate the Caspase 8 Substrate working solution plate at room temperature for 30 to 60 minutes, protected from light. Note: If desired, add 1 µL of the 1 mM Ac-IETD-CHO caspase 8 inhibitor to selected samples 10 minutes before adding Caspase 8 Substrate working solution at room temperature to confirm the inhibition of the caspase 8-like activities.
   
   
5. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).
   
   
6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 370/450 nm (Cutoff = 420 nm).