Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red 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 1 µL of 500X Nuclear Red™ CCS2 into 0.5 mL of cell solution
3. Incubate at 37 °C, 5% CO₂ for 10 - 30 minutes
4. Analyze cells using flow cytometer with PE-Texas Red channel (Ex/Em = 488 nm/615 nm)

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

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.
   
   
2. Treat cells with test compounds for a desired period of time to induce apoptosis, cell cycle arrest or other cell cycle functions.
   
   
3. Add 1 µL of 500X Nuclear Red™ CCS2 (Component A) in cells containing growth medium.
   
   
4. Incubate the cells in a 37 °C, 5% CO₂ incubator for 10 to 30 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 Red™ CCS2. The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. It is not necessary to fix cells before staining since Nuclear Red™ CCS2 is cell- permeable.
   
   
5. Optional: Centrifuge cells at 1000 rpm for 4 minutes and re-suspend cells in 0.5 mL of assay buffer (Component B) or a buffer of your choice.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with PE-Texas Red channel (Ex/Em = 488/615 nm). Gate the cells of interest, excluding debris.