Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit *Blue Fluorescence*

1. Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate).

2. Add an equal volume of Caspase 3/7 Substrate working solution.

3. Incubate at room temperature for 1 hour.

4. Monitor fluorescence intensity at Ex/Em = 360/470 nm (Cutoff = 420 nm).

Thaw one vial of each kit component at room temperature before starting the experiment.

For guidelines on cell sample preparation, please visit:

https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Prepare a Caspase 3/7 Substrate working solution by combining 50 µL of Caspase 3/7 Substrate (Component A) with 10 mL of Assay Buffer (Component B), and mix well.

   Note: Aliquot and store any unused Components A and B at -20°C. Avoid repeated freeze/thaw cycles.

1. To treat cells, add 10 µL per well of 10X test compounds for a 96-well plate or 5 µL per well of 5X test compounds for a 384-well plate into PBS or the desired buffer. For blank wells containing only medium, add an equivalent volume of the compound buffer.

2. Incubate the cell plate at 37°C with 5% CO2 for a desired duration to induce apoptosis (e.g., 4 to 6 hours for Jurkat cells treated with camptothecin). Adjust the time as necessary based on the cell type and treatment conditions.

3. Add 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate of Caspase 3/7 Substrate working solution.

4. Incubate the plate at room temperature for at least 1 hour, protected from light.

   Note: To confirm the inhibition of the caspase 3/7-like activities, add 1 µL of 1 mM Ac-DEVD-CHO caspase 3/7 inhibitor to selected samples 10 minutes before adding the Caspase 3/7 working solution at room temperature.

5. Centrifuge cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes (brake off).

6. Monitor the fluorescence intensity with a fluorescence microplate reader at Ex/Em = 360/470 nm (Cutoff = 420 nm).