differentiated PC12 cells detatching - (Jun/24/2007 )

Hi All,
I have PC12 cell cultures which I stably transfected a while ago and they are growing nicely. I differentiated them using NGF(50ng/ml) and culturing in low-serum media and they looked beautiful. I then washed them with PBS as part of immuno expt and they all (at least 80%) detached-not happy . I had coated my coverslips with Poly-L-Lysine and when adding media/wash steps always touch the pipette tip to the wall of the well so as not to detach the cells. This is only happening with my differentiated PC12s. What should I try ?(I am running out of time quickly so am very desperate!)
Thanks for any suggestions

-daph-

QUOTE (daph @ Jun 24 2007, 07:41 PM)

. I had coated my coverslips with Poly-L-Lysine and when adding media/wash steps always touch the pipette tip to the wall of the well so as not to detach the cells. This is only happening with my differentiated PC12s. What should I try ?(I am running out of time quickly so am very desperate!)
Thanks for any suggestions

try using PBS with calcium and magnesium. It can prevent detaching. Use it to make all solutions for immunostaining.

-scolix-

QUOTE (scolix @ Jun 25 2007, 10:27 AM)

try using PBS with calcium and magnesium. It can prevent detaching. Use it to make all solutions for immunostaining.

Thanks for the advice, what concentrations of calcium and magnesium should I use?

-daph-

QUOTE (daph @ Jun 25 2007, 05:31 PM)

QUOTE (scolix @ Jun 25 2007, 10:27 AM)

try using PBS with calcium and magnesium. It can prevent detaching. Use it to make all solutions for immunostaining.

Thanks for the advice, what concentrations of calcium and magnesium should I use?

We buy 10x DPBS with calcium and magnesium from Invitrogen and dilute it to 1x and adjust pH.

You could check their website for the concentrations.

-scolix-