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

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 3/7 Substrate working solution
3. Incubate at room temperature for 1 hour
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

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

Add 50 µL of Caspase 3/7 Substrate (Component A) into 10 mL of Assay Buffer (Component B) and mix well to make Caspase 3/7 Substrate working solution.

Note: Aliquot and store the unused Caspase 3/7 Substrate (Components A) and Assay Buffer (Component B) at -20 ^oC. Avoid repeated freeze/thaw cycles

• For adherent cells: Plate cells overnight in growth medium at 20,000 cells/well/90 uL for a 96-well plate or 5,000 cells/well/20 uL for a 384-well plate. 
• For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 80,000 cells/well/90 uL for a 96-well poly-D lysine plate or 20,000 cells/well/20 uL for a 384-well poly-D lysine 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.

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 (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.

3. Add 100 µL/well (96-well plate) or 25 µL/well (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: If desired, add 1 µL of the 1 mM Ac-DEVD-CHO caspase 3/7 inhibitor into selected samples 10 minutes before adding Caspase 3/7 working solution at room temperature to confirm the inhibition of the caspase 3/7-like activities.

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 = 490/525 nm (Cutoff = 515 nm).