Hi Ioannis -

This looks like a very nice histogram to me.  You don't give the actual MFI's of the peaks, so I have to judge by eye.... The G0 peak has an MFI of about 195 and the G2 peak is about 390 giving a ratio of 1.95.  Perfectly acceptable!  Peak resolution looks good also.  You are correct - G2/G0 ratio ideally is, by definition, 2.0.

You don't give much specific info about PI and cell concentration, incubation times, cytometer used, or how you are gating/defining the G0 and G2 peaks, etc.  I honestly don't think you are going to improve on your staining protocol.  Perhaps your gating protocol is the issue?

I agree with Lynda. Your G1 peak looks slightly shifted to the left of 200, try adjusting your parameters to shift the peaks slightly to the right. That will also separate the peaks more and provide data that is a little easier to judge by eye. Although without knowing the actual MFI's I can't be certain about the separation. 

Good Luck.

Well, if I get peaks like these I would completely accept them. They look real good!

Anyway for reference, if you add RNAse A it could improve the resolution if that is what you are after.

Hi there. Thank you very much for your answers.

Well the MFI values are 196 and 353 so the ratio is about 1.8.

Gating is performed first on a FSC-SSC plot and then doublets are excluded in a FL2A-FL2W plot. This is actually a test experiment with 10,000 events in the gate set on the FSC-SSC plot on a BD FACSCalibur cytometer. I cannot answer on the PI and cell concentration at the moment, as I am not the one doing the staining, but I will look to it. 

Regarding the way to define the peaks, I am using the automated function of FlowJo, which adjusts two normal curves on the peaks. Do you think it would be better to do it by eye?

Thank you again all.

I beg to differ with Florian, but the automated models, unless you have very strange cells, are the best way to go when looking at single color cell cycle histograms.  There is simply too much overlap of the G0-S peaks and S-G2 peaks- even in the best of cases.  Using the old style 'half peak height' manual gating method to define G0 and G2 is extremely subjective.  Stick with FlowJo's models.

I think Lynda has covered it. Your peaks do look good, and 1.8 is acceptable.  

1.  If you are using ethanol fixation, it is best to fix at least overnight.

2.  Also so as to ensure that you have saturated all DNA binding sites with PI, and PI has entered all DNA sites, PI at ~40 ug/ml with staining for minimum 1hr is recommended.  I generally, leave PI staining for at least 3-4hrs and most often overnight.  This overnight step gives nice sharp peaks (realtively speaking, of course), since all cells are stained to saturation.  This also helps delineate G1 vs G2 peaks better.  G2 cells have more condensed DNA (tighter packing of DNA-chromatin) than G1 cells, and so dye penetration in G2 cells is not equivalent as G1 cells. So in practice, even under best conditions G2/G1 ratio is

Another question about the cell cycle analysis by using Flowyjo:

how about the constraints? Do you generally set them or perform an unconstrained analysis?