Did you include a control with a high apoptosis rate (like cisplatin or TRAIL treated cells) ? Is it possible that your apoptosis rate is low and doesn't allow you to see the peak for your apoptotic cells ? Did you check the apoptotic phenotype of your cells under the microscope ? 

Another explanation could be the flow speed you used when you scan your cells. You need to use the slowest flow ta have a better resolution of the PI signal.

To measure apoptosis by FACS analysis, I would recommand to use Annexin V or cleaved Caspase 3 detection as they are more sensitive.

Delannoy, thank you for your suggestion.

Cells i used were 7 days cultured cells, so apoptosis rate was high.

And i checked lots of cells were dead before i preparing the experiment.

I set high flow rate when i scan my cells, so i think it can be problem.

Your suggestion was so helpful to me

 Thank you 

If you use high flow rate you will have doublets, triplets etc. which will effect your results. Low flow rate is the best for DNA analysis. If you are doing this test only for screening, it is acceptable, but usually you need to confirm your apoptosis results with another method such as Annexin V/PI, TUNNEL etc. 

Jihun,

I have followed the protocol and it comes fine. However, you may not get a sharp G1 peak always, it may be slightly broad also. Check Counts vs FL2A histogram and see whether you are getting  proper apoptotic population. Gate your cells on FL2A vs FL2W dot plot to choose the correct population for analysis. Also , check your reagents for sample preparation. The DNA extraction buffer should have pH 7.8. Also as suggested, always work with low flow rate for DNA analysis experiments.

All the Best