Cell Cycle Analysis - crucial to keep cells cold after ethanol fixation, and aft - (Oct/07/2014 )

I have been processing cells for cell cycle. I would like some clarification on the processing temperature requirements (i.e. sample and reagent temperatures) for the following: 

1. After the cells have been ethanol fixed for a period of time at -20C, is it necessary to keep the cells on ice during the processing for PI staining (PBS wash)? Does the PBS need to be cold?

2. After PI staining is it necessary to keep the cells on ice? I perform the PI staining at RT. 

3. What reagents need to be kept ice cold? Is RT PBS fine or should I be using ice cold for washing?

I am only keeping the 70% ethanol ice cold, and I am only keeping the cells cold when I place them in the freezer after ethanol fixation. Washing steps are done with RT PBS. I am just wondering if there are some steps after the ethanol fixation where keeping the cells cold is crucial. Would this be important for keeping the cells from lysing or clumping?

Thanks for any feedback you can provide!

Once fixed the cells should be fine at room temperature. You shouldn't need to stain with PI or do your washes with cold PBS. On the other hand, low temperatures can increase the stringency of antibody binding, so it can be useful to do antibody binding steps on ice.