3 replies to this topic

[EGF]

Hi,
I am trying to do IF with neuroblastoma cells (SK-N-SH) using our "traditional" protocol, which works fine with HEK, COS and so on.
We plate cells on 12-well plates with a sterile, poly-lisinated coverslip inside, and then perform transfection or IF.
Usually when we plate the cells on such coverslips, cells grow well and only the cells affected by the movement of the coverslip around the well die.
My neuroblastoma cells look healthy when plated on larger flasks with good confluence, but when I tripsinize and plate them for IF, occurs a massive death and surviving cells look terrible. If I use non-lisinated coverslips, it is worst. Transfection efficiency with lipofectamine is poor.

What can I do? I try not to plate them at too much low confluence because they are very sensible.

[labrat612]

Do you coat your coverslips with something like Poly-L-lysine? That might help. I used to work with N2a cells, and we did that routinely. It didn't interfere with transfection or IF.

[EGF]

labrat612, on Mar 30 2010, 08:32 AM, said:

Do you coat your coverslips with something like Poly-L-lysine? That might help. I used to work with N2a cells, and we did that routinely. It didn't interfere with transfection or IF.

Yes, the coverslips are coated with poly-L-lysine...

[labrat612]

Your cells may respond to poly-D-lysine better. Or even another type of matrix. I would research your cell line via research articles and see how other labs have handled your cell line.