Why my cells dont grow - hippocampal cell (Sep/18/2006 )

I am doing hippocampal cell culture in neurobasal+ B27. but my cells dont grow . What can be the reason?

there could b too many reasons, starting from the different solutions to the the way u handle the cells.

u need to coat the plates, as these help and getting the right substrate for cell growth differs.

the time taken for dissection of the tissue and and further handling can make a difference.

plating them in an optimum density is also important.

these r just a few. but infact every single step could make the difference.

if u could give a detailed description of the preps, we could try trouble shooting.

I use CMF-HBSS. coat my plates with l-lysine ( I used it with two concentrations 0.1% and 0.01% according to some papers). I cut their heads and put them in CMF media, remove their brains and put their brains in another pettri dish containing CMF and after that nearly within 3-5 min lasts to me to isolate the hippocampi under a loop in a petri dish containing CMF media. I put them in trypsin 0.25% for 10-15 min (every 10 hippocampus in a falcon tube) and triturate them with a fire-polished pasteur pippet (10-15 stroke). Then centrifuge the supernatant in 900 g for 3 min. And put 100000 cell in every 1 cm2.
Do you think my protocol does have any problem?

And another thing that I am thinking about is my incubator. My inc. is new and I am the first person who is using it. How I can check my incubator?

a fyrite tester for CO2 levels is a good idea? as well as a thermometer to check the temperature calibration, at least with the initial set-up

I use CMF-HBSS. coat my plates with l-lysine ( I used it with two concentrations 0.1% and 0.01% according to some papers). I cut their heads and put them in CMF media, remove their brains and put their brains in another pettri dish containing CMF and after that nearly within 3-5 min lasts to me to isolate the hippocampi under a loop in a petri dish containing CMF media. I put them in trypsin 0.25% for 10-15 min (every 10 hippocampus in a falcon tube) and triturate them with a fire-polished pasteur pippet (10-15 stroke). Then centrifuge the supernatant in 900 g for 3 min. And put 100000 cell in every 1 cm2.
Do you think my protocol does have any problem?

And another thing that I am thinking about is my incubator. My inc. is new and I am the first person who is using it. How I can check my incubator?

Thanks a lot for your helps. Finally yesterday I understood what is my problem (after about 45 day). The problem was my poly l-lysine. I did not notice that the l-lysin which I used had conservative material. It is so stupid! but I learnt many things during those days!