Cell Meter™ Nuclear Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Add 5 µL of 200X Nuclear Green™ DCS1 into 1 mL of cell solution
3. Incubate the cells in a 37°C, 5% CO₂ incubator for 30 to 60 minutes
4. Pellet the cells and resuspend the cells in 1 mL of growth medium
5. Analyze the fluorescence intensity using flow cytometer with FL1 channel

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

1. For each sample, prepare cells in 1 mL of warm medium or buffer of your choice at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
   
   
2. Treat cells with test compounds for a desired period of time to induce apoptosis.
   
   
3. Add 5 µL of 200X Nuclear Green™ DCS1 (Component A) into the treated cells.
   
   
4. Incubate the cell solution in a 37°C, 5% CO₂ incubator for 30 to 60 minutes. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intack, and wash the cells once with serum-containing media prior to the incubation with Nuclear Green™ DCS1 dye-loading solution. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
5. Optional: Centrifuge the cells at 1000 rpm for 4 minutes, and then re-suspend cells in 1 mL of Assay Buffer (Component B) or buffer of your choice.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/525 nm). Gate on the cells of interest, excluding debris.