Dear Kaipol,

It is so clear you have subpopulation in your results, i.e., apoptotic, necrotic or cell debris as you mentioned, which may indicate you treat your cells with something that lead to these effects, or u may induced it within your procedure such as harvesting or centrifuging the cells. If not of these, try to carefully adjust your setting parameters when u run the flow cytometry machine.

Good luck

 Thank you. Just a little more, would you discard those apoptotic population in cell cycle assay.

What instrument and threshold levels were used in the experiment? To me it looks like debris, apoptotic cells are typically located closer to G1 cells. You can try to increase threshold and gate-off debris, apoptotic nuclei have to be counted.