Hei !
Im using mouse endothelial cell line for practicing cell sorting by FACSAria. The problem is, that adharent cell line is not anymore adharent after sorting procedure. Im using size 100 nozzle, coat 25 size flasks with 0.2% gelatine and using rich media (20% serum, 1% AA). After sort cells are alive. Lasers are blue and red. Can anybody help ?

Faust

Have you checked viability the next day after sort? Perhaps their membranes were intact after sort but something else had started to kill them? Were they left with antibodies with NaN3 for too long at a high tmeperature?

I havent check with Tryphan blue, just visually. Next time I will try. Antibody staining lasts 20-30 min on ice, after that Im keeping cells all the time on ice.

Is it needed to remove bound antibody from cells to get them grow happily and adharently ? How to do it ?

I would use trypan blue to count viable cells to at least see if they are alive to start with. You could also use propidium iodide (PI) and resort a small sample of your sorted cells and check for viablity at the same time. ARe you using PI during the sort? If no, you may want to use it, that way you sort only viable cells.

Your antibody staining sounds just fine and I really can't imagine that the antibody is interfereing with adherence.

Maybe your sorting at too high of pressure for your cells. Check that. No chance that the sheath fluid is homemade and somebody added too much or too little water?

Problem solved with higher serum concentration in sorting buffer.

Congratultions and thanks for sharing your successful solution!