HELP! CORTICAL NEURONS - (Jan/10/2006 )
My cortical cultures (from rat fetuses) have a lot apoptotic cells, some trash and chunks. I would like to optimalize the procedure including media, minceing, digestion et cetera. Did you get any information on this subject? I would be greateful for any help. I used different procedures, including those from protocol-online, well the effect wasn't satisfying.
well, it's somewhat difficult to answer your question without knowing the details (day of pregnancy, rat strain, culture media, plating density, cell culture substrate and the coating technique; finally, what are you going to do with the neurons?).
Some basic things:
1. Cortical neurons will start dying after a week in culture unless you co-culture them with astrocytes
2. removing cell debris just after the brains were homogenized (i.e., before plating the cells) improves plating efficiency
3. plating density seriously affects the viability of cultured neurons
4. coating technique is essential
5. it’s really important to change media carefully, avoiding drying of cells. The best way is to change half of the volume of media each time
good luck,
mikhail
My cortical cultures (from rat fetuses) have a lot apoptotic cells, some trash and chunks. I would like to optimalize the procedure including media, minceing, digestion et cetera. Did you get any information on this subject? I would be greateful for any help. I used different procedures, including those from protocol-online, well the effect wasn't satisfying.
4. coating technique is essential
mikhail
I do corical cultures from rat fetuses as well. So far, my cells are not consistent. Sometimes they spread out individually (great), but sometimes they clump up as clusters. It looks like the coating problem. I coated with poly-l-lysine hydrobromide (high mol. w. pll with enhanced solubility) at 20 ug/ml (higher conc. appeared to harm cells) overnight and washed with water twice. Some people mentioned laminin coating on top of that, I tried (10 ug/ml in media, 1hr, washed with media, and dried) but had more cell clustering.
Can anyone give suggestion about a coating technique that is consistent with cell spreading (not clumping)?
min
>4. coating technique is essential
I use this protocol http://www.cambrex.com/Content/Documents/B...%20Cultures.pdf
Poly-L-lysine is not good for neuronal cultures because neurons (in contrast to astrocytes) tend to cleave poly-L-lysine - that's why cultured neurons aggregate with time. Poly-D-lysine, or Poly-L-lysine+laminin coating works fine.
You can also try commercially available poly-D-lysine/laminin coated coverslips to troubleshoot your culturing. Think about proper coverslips too. Some brands of glass surfaces are better then others. I use Fisherbrand culture coverslips from Fisher Sci.