Hello
I'm trying to evalute the cell cycle in a lung cell line (NL-20) by flow cytometry I'm new in this type of experiments. I have used PI and DRAQ5 fluorocrhomes, but i only get 1 peak in my histogram and not the 3 peaks that usually apear in this kind of experiments.
I dont know if it is because I made a bad gating and i'm losing the signal or because i need to change something in my protocol. Do you have any sugestion of what I have to do?
Keep in mind you need a single cell suspension first, and then you have to vortex the cells while you slowly add fixative agent drop to drop. Some cells tend to group and they are fixed as groups. You need to obtain a single cell fixated suspension of cells in order to able to identify different pikes, groups of two cells will interfere with your experiment. That is one possible technical problem, the other thing to consider is that since your cells are not normal cells sometimes different populations with different karyotypes coexists making this kind of experiment hard to analyses.
It's hard to answer your question without seeing how the data look like.
it can be
1. problem in sample preparation.
2. problem in gating
3. problem in the aquisition
How do your FSC/SSC and PI/FSC plot look like?
I agree with Diego Raffo and Yoav Lubelsky . There might be various reasons for occurrence of single broad peak. It will be difficult to answer your query without looking at your plot output.
- Badges
- Science method