Cell cycle and Hoescht Stain

[apelge]

Hi all,
I have a question:
I am trying to optimise a protocol to analyse the cell cycle in some melanoma cells after treatment.
I am working with a BD LSRII (brand new) so I started to see how the machine was working.
After a little work with the software i started to obtain the first results.
I am staining my cells with hoescht , without fixation and permeabilization.
I am using 1 ug/ml of stain, and i wait 30’ before the analisys.
Now, the problem is that suddenly i cannot reproduce what i was obtaining, with any of the cell lines I was using!!!I tryed two different hoescht staining with different ability to permeate the cell, but nothing...
I do not obtain the classical cell cycle pattern anymore but just one huge peak at low fluorescence.
May I ask if anybody has some suggestion to give me?
I cannot fix and permeabilise the cells, and they are in complete medium…