Well, you could try to detach the cells by a solution with higher EDTA concentration, but I doubt that HeLa can not be trypsinesed.

Did you try a simple, idiotic  tapping of the flask? I usually put trypsine on the cells, put the flask back in the incubator for about 2-3 minutes and then lightly tap with my palm around the flask. They always detach.

Dear Ljerka,

Yes It is strange, caz my other cells nicely detaching with my trypsine only HeLa is not responding. Yes I do tapping the flask to detached cells, even after that Hela stay still attached to the flask.

There is a recipe for detaching primary macrophages from surface.  It should work even for the most "stubborn" cel lines.

It's. PBS + 3mM EDTA + 10 mM Glucose

It has to be sterile, but you can use it in the sam way as trypsine.

do you make your own trypsine? Perhaps it's too weak?

We usually remove the medium from cells, washed by 1x PBS and Trypsin-EDTA followed by Trypsin -EDTA addition and incubated in CO2 incubator for 2-3 minutes (3-5 minutes for stubborn cells). The cells are finely detached. If not, a gentle tapping of the plate/flask or using cell scrapper will aid detaching. Sometimes, serum may inhibit Trypsin action. This can be rectified by rinsing cells with PBS. Please find the attachment. 

Also, passage number of the cells may remain as one of the factors affecting detachment via trypsin action.

I make my own trypsin, but the procedure is same as yours. Serum is trypsin inhibitor, so growth medium must be removed, but I just can not figure out why the cells won't be released from the surface. 

Thanks a lot for your comments and ideas. Ill try it

Cheers

HeLa gets very much attached to the surface of dish. for 10cm dishes, I use 2ml trypsin-EDTA and put them in the incubator for 3-4 mins. they need a little extra tap. haha, so try tapping them for a bit. i have seen people using cell scraper - if trypsin is not working for your cells maybe that's something you could try