Cell Meter™ Caspase 8 Activity Apoptosis Assay Kit *Red 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 1 hour
4. Monitor fluorescence intensity (top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm)

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

1. Ac-IETD-ProRed™ stock solution (200X):
Add 65 µL DMSO into the vial of Ac-IETD-ProRed™ (Component A) and mix well to make 200X Ac-IETD-ProRed™ stock solution. Protect from light.

Add 50 µL of 200X Ac-IETD-ProRed™ stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 8 Substrate working solution. Protect from light. Note: Caspase 8 working solution is not stable, use it promptly.

Prepare cells:

• For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well or 5,000cells/well/20 uL for a 384-well plate black wall/clear bottom plate.
  
  
• For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 to 200,000 cells/well/90 uL for a 96-well or 20,000 to 50,000 cells/well/20 uL for a 384-well black wall/clear bottom plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

Sample Protocol:

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 37°C, 5% CO₂, incubator for a desired period of time (3 - 4 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 Caspase 8 Substrate working solution.
   
   
4. Incubate the plate at room temperature for at least 1 hour, 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. Monitor the fluorescence intensity with a fluorescence microplate reader (either top or bottom read mode) at Ex/Em = 540/620 nm (Cutoff = 610 nm). 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.