Hi,today I did the same with 3T3 but in 6 well plates I put 20000 cells in order to have tomorrow afternoon 60% (12hOO) necessary for the transfection.so I will have the same problem lets return to your problem, I will trypsine just 1min,37°c,1ml trypsine or wait 12h (tiime necessary for having 60% cells )tomorrow and than contes the cells and finally put 5000 cells/well don't worry 3T3 are very resistant cells but what you try to express exactly? if is not confidentiel.

I am trying to express melanopsin (opsin of ganglion cells) with a CMV promoter. It is causing the plasma membrane to be very studded with proteins and the cells to be very grainy looking. Are there any other techniques that don't involve trypsinizing the cells?

The point of trying to keep the individual colonies separate is that they may have individual characteristics that differ from each other. This is exactly the same when you transform bacteria and pick individual separate colonies - you never mix two different colonies together. In your case, your gene of interest may be differentially expressed in each colony. An alternative to trypsinization is a cell scraping technique - there are disposable instruments available to do this, and your tissue culture facility might already have them. I would keep them as separate as possible, plate into individual wells, and then screen for expression.