Like people, cells don't like to be alone. Have you tried plating them in conditioned medium (ie. the medium the cells were in before passaging)? That might improve survival. 

Dear Virlana,

I think Karl is right. We had similar problems with our cell lines that don't like to grow alone. You can try the approach with conditioned medium. In case this is also not working (it was not working in our hands), you can also seed the cells in a low confluence in a 10cm dish, wait for colony formation and then pick the clones and transfer them to 96 well.

Thank you both for your quick replies! I have not tried conditioned media, good suggestion! What's disconcerting is that our collaborator has plated cells in both wells of 96-well plates as well as 10 cm dishes, and even untransfected WT cells that have gone through the sorter and are in the 10 cm dishes are growing, but not well.

Hello Virlana,

I worked with fibroblasts cells, I did the KO in bovine TFAM gene. I used many protocols, the protocol that work better was supplement the culture medium with more fetal bovine serum. I used 10% SFB.

First I did the sorting of the cells and I did the clones  in 96 wells plate, after 10 days I split the clones in 24 well/plate but the clones not grew than I changed I used the conditioned medium and after sorting in 96 wells I waited 10 days approximately, depends on the cell and I split in 6 well plate direcly and worked.

I change the conditional medium every two days.

mammalian cells generally have a hard time growing without the appropriate cell density. That especially goes for adherent cells. They are supposed to grow in a solid tissue, so sitting around alone makes it hard for them. 

If you only care for the GFP+ cells and can effort a second round of sorting, I would suggest to co-incubate the single clones with non fluorescent cells (or stained differently). Let the cells grow out and sort again for the GFP+ cells.