Cell Meter™ Caspase 9 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 9 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.

Add 5 µL of 200X Ac-LEHD-ProRed™ stock solution (Component A) into 1 mL of Assay Buffer (Component B) and mix well to make Caspase 9 Substrate working solution. Protect from light. Note: Caspase 9 working solution is not stable, use it promptly. 1 mL of Caspase 9 Substrate working solution is enough for 10 assays. Note: Aliquot and store unused caspase 9 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- 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-wel or 25 µL/well/384-well plate of Caspase 9 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-LEHD-CHO caspase 9 inhibitor to selected samples 10 minutes before adding Caspase 9 Substarte working solution at room temperature to confirm the inhibition of the caspase 9-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 non-adherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.