Adult rat dorsal root ganglia neurons - (Apr/16/2010 )

I do not understand why my primary culture of adult rat dorsal ganglia neurons at times attach at the substrate and at time they float in the media. I set up the protocol for the isolation. They are perfect before the plating. I treated dish with poli-D-lysine (high molecular weight)(20microgram/ml in HBSS without Ca/Mg) and laminin (18microgram/ml in HBSS without Ca/Mg ) for 24 hours in the incubator and I wash with water. I let dry the dish and then I plate the neuron in a drop and after 2 hours I feed with media (F12, L-glutamine 2mM, heat inactivated FBS 10%, Pen/Strep, Fungizone)
Any suggestions?????

gabriella on Apr 16 2010, 05:20 AM said:

In my experience, not all of the neurons will attach to the slip, no matter what you coat with. Your plating protocol sounds good, but you might try using Neurobasal-A supplemented with 2mM L-Glutamine, 10ng/mL NGF, B27 serum replacement and Pen/Strep instead of F12.

Also, how old are your rats? Younger animals are generally easier to culture.

Thank you for the information. My rats are 8 weeks old. What do you mean with B27 serum replacement. I have to add B27 and no serum. But how much B27?
Thanks. Gabriella

gabriella on Apr 19 2010, 03:24 AM said:

B-27 is a culture supplement used instead of serum. FBS can have significant variation between lots. Most people get it from Invitrogen. Here is the product page. It's supplied as a 50X concentrate, just add enough to the medium to get a 1X concentration. Using B-27 instead of serum helps cut down on proliferation of glia and fibroblasts in long-term cultures and can help improve viability of neurons.