3 replies to this topic

[gabriella]

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?????

[gfischer]

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

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?????

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.

[gabriella]

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

[gfischer]

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

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

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.