Dissociation of HaCaT cells without using trypsin - will PBS/EDTA work? - (Jan/16/2006 )
We are planning some experiments involving HaCaT cells, and we will have to dissociate them from their growth flasks. However, we have to keep receptors in the membranes intact so trypsination is not an option. We don't want to use a cell scraper either since that may cause damage to a rather high number of cells.
Has anyone used PBS with EDTA (or EGTA) to do this? If so, how long do you have to incubate and what concentration of EDTA (or EGTA) have you used? Single cell suspensions is not of importance here.
Hi Sonjaa, I don't have any experience here but have been looking to do the same thing. One protocol I found used PBS 0.02% EDTA and incubated on ice for 15 minutes. I'm guessing that the timeing will vary with cell type. I'm looking forward to hear what others say. Jon
I'm going to try a product called Accutase today, it's supposed to be very gentle and not disrupt surface antigens. I wont be looking at any surface proteins today but plan on using it for flow analysis on primary macrophages in the future. I'll let you know how I make out. Here's a link to some info. http://22.214.171.124/search?q=cache:kyadF...lient=firefox-a
Sounds interesting, I'll be happy to hear how it works. We don't have our HaCaT cells up and going yet, but I guess I'll start them in a week or so. Then I'll test them and see how the PBS/EDTA works. I'll let you know.
well I just started working with HaCat (this Jan). Initially they were growing fine but after three months I found that suddenly after splitting my cells the next day most of the cells are dead and are seen floating.
I tird growing fresh cells from frozen stock but.. even they never grew.. so I have thawed another stock culture... and its taking its own sweet time... to grow... i am not even sure if it would make it..
Have you guys faced any such problems.. could suggest something to help me out...
Thanks a lot
Hope to hear from you guys soon