Cell Meter™ Fluorimetric Fixed 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. Fix cells with 70% Ethanol
3. Add 5 µL of 100X Nuclear Red™ CCS1 and 5 µL of RNase A into 0.5 mL of cells solution
4. Incubate at room temperature for 30 - 60 minutes
5. Analyze cells using a flow cytometer with FL2 channel

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

1. Treat cells with test compounds for a desired period of time to induce apoptosis or other cell cycle functions.
   
   
2. For each sample, prepare cells in 0.5 mL PBS at a density of 5 × 10⁵ to 1 × 10⁶ cells/mL. 
   
   For Adherent Cells: The cells are trypsinized, suspended in 10% FBS medium, centrifuged (1000 rpm, 5 min), and the pellets are resuspended in PBS.
   
   For Suspension Cells: The cells are centrifuged (1000 rpm, 5 min), and the pellets suspended in PBS (1 mL). Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.

Fix the cells with 70% Ethanol: 

1. Pipet 0.5 mL cell suspension into 1.2 mL absolute Ethanol (final concentration approx. 70%).
   
   
2. Incubate cells on ice for at least 2 hours (or overnight at -20°C). Cells can be stored at -20°C for up to 2 years before staining. Note: Ethanol is commonly used for fixation after cell surface antigens were stained with monoclonal antibodies, while methanol is commonly used for fixation after intracellular antigens were stained with monoclonal antibodies. In this procedure whole cells are fixed and analyzed. Because the entire cell mass is still present, the use of RNase is typically included to eliminate any double-stranded RNA. Despite the fact that whole cells are being analyzed, attempts to detect some intracellular antigens in conjunction with DNA may fail because the proteins leak out of the permeabilized cell (e.g. green fluorescent protein). In these cases a brief pre-fixation (10 minutes at 4 - 6°C) with 1% paraformaldehyde in PBS before the alcohol fixation will help retain the proteins in the cell.

Stain the cells with Nuclear Red™ CCS1:

1. Pellet the cells at 1000 rpm for 5 minutes and wash cells at least once with cold PBS.
   
   
2. Suspend the pellet in 0.5 mL of Assay buffer (Component C).
   
   
3. Add 5 µL of 100X Nuclear Red™ CCS1 (Component A) and 5 µL of 100X RNase A (Component B).
   
   
4. Incubate the cells at room temperature for 30 to 60 minutes. Note: 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 5 minutes, and re-suspend cells in 0.5 mL of assay buffer (Component B) or buffer of your choice.
   
   
6. Monitor the fluorescence intensity with a flow cytometer using FL2 channel (Ex/Em = 490/620 nm). Gate on the cells of interest, excluding debris.