I've made a few attempts at flow cytometry using RPE-1 cells (ATCC), but have been getting issues with colony formation. This problem exists even for untransfected cells at the slowest sorting speed on the BD FACSAria II. After sorting single cells into a 96-well plate, I'll sometimes find what looks an enormous non-dividing single-cell - this is definitely not the parent fibroblast-like phenotype.

I've tried doing serial dilutions of these cells to see if I can replicate the slow/no growth phenotype, and I definitely slow growth with fewer cells, but they do grow, eventually. I have not been able to replicate that enormous non-dividing cell phenotype, though.

For those who have done single-cell sorting with this cell type, are there certain conditions that are critical for colony formation? What conditions have you been successful with?