Hi Sharon, 

Let's assume that your 4 hours start from after you finished all the staining procedure. Which means that including your staining and cell prep time, it took about 6 to 7 hours in total? FACS buffer contains a very limited amount of FBS, I will say it's not really a good condition for unfixed cells to maintain their physiological condition.

First, you may want to check whether the SSC and FSC changed. I believe you will find more debris (small SSC and FSC portion increase). These will be dead cells.If most of your cells were dead, it is possible to get a significant MFI signal decrease.

p/s: Did you avoid light during these on ice time?  

Thanks Shulin. Yes, I left the cells on ice in the dark.

What you suggested might be true, but in my positive control, the MFI signal remained high and was consistent with what I saw in several experiments before....(unless the signal was originally so high such that any amount of time on ice didn't make a difference...)

I think It depends on your staining protocol and type of cells. and if you use lysing solution 

eg while I measure HSC cd 34 using stem cell kit

if I.use microspheres   , the viabilty  and subsequently the MFI , was decreased by time  only 1 hour ia allowed as the manufacture said 

Hi everyone, 

I had an experience with human Lymphocytes. It was 6-8 hour between isolation and FACS analysis (no fixation, cells were in PBS 1% human serum, on ice, protected from the light).

Results were as usual. So it is possible in case of extreme need but of course better to acquire asap.

Good luck!