Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*

Protocol summary

1. Prepare cells with test compounds at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL
2. Add 1 µL of 500X Nuclear Violet™ into 0.5 mL of cell solution
3. Incubate at room temperature for 30 - 60 minutes
4. Analyze with a flow cytometer using the 405 nm violet laser excitation

Important notes
Thaw all the components at room temperature before use.

1. For each sample, prepare cells in 0.5 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 or other cell cycle functions.
   
   
3. Add 1 µL of 500X Nuclear Violet™ (Component A) into the cells containing the growth medium, and incubate the cells 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 intact, and wash the cells once with serum-containing media prior to incubation with Nuclear Violet™. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. Note: It is not necessary to fix the cells before DNA staining since the Nuclear Violet™ is cell-permeable. Note: Human and rodent stem cells efflux Nuclear Violet™ stain, and this is the basis for the Side Population (SP) technique; the efflux can be blocked with blocking agents like verapamil, fumitermorgin C to prevent dye efflux for accurate DNA content analysis.
   
   
4. Optional: Centrifuge the cells at 1000 rpm for 4 minutes, and then re-suspend cells in 0.5 mL of assay buffer (Component B) or the buffer of your choice.
   
   
5. Monitor the fluorescence intensity by flow cytometry using the 405 nm violet laser (Ex/Em = 405/445 nm). Gate on the cells of interest, excluding debris.