I am doing culture of hippocampal cell in coverslip coated with 1mg Poly D lysine/ml 0.1M boric acid solution (PH 8.5) but now a days its not working fine as what could be the problem. Usually first we prepare 10mg/ml stock and store in -20 C then we warm in 37 C water bath and mixed with boric acid for final treatment to coverslip and keep it for 2 days at room temperature and after washing with Q water left it in medium containing well for another 2 days. But the cell now a days not growing well. we seed 142000 cells/12mm coverslip in Macs neuro medium containing well.
We use Poly-L-Lysine at 10-20ug/ml in PBS/HBSS for 1-2 hours at 37 C to coat the cell culture dishes or covers slips. After that we wash it 3 times by rinsing in PBS/HBSS.
If it doesn't work you may use some other ECMs like Fibronectin or Laminin to coat the surface.It works well.
May help you
Hi!
It's always frustrating when neurons have been growing well and then are not. Off hand, I don't see anything wrong with your coating protocol, as long as you rinse the coverslips well before seeding the cells. Having said that, I usually only use that much Poly-D-Lysine in borate buffer if I am using it to link ECM proteins to the glass. Depending on the age of the neurons, I would use a more dilute solution in water. High pH borate buffer causes the Poly-Lysine to coil more tightly into an alpha-helix, so that you get more charge per area. Sometimes that is too much charge, and the neuronal processes actually have a more difficult time growing on the substrate because it is too "sticky"
Unfortunately, I don't know what you mean specifically by the neurons "not growing well", so I can only give general troubleshooting suggestions. Since your neurons were doing fine up to this point, the first thing I would suggest is that you check the age and expiration date of the media, the supplements (including the glutamine), and the Poly-Lysine. Sometimes the neurons are very fussy, and if the supplements or media are near the expiration date, they start to grow poorly.
Another possibility is the coverslips themselves. Have you recently purchased a new batch of coverslips? Did something change about the cleaning protocol, or how they were sterilized?
Lastly, are the same people preparing the dissociated neurons by the same protocol? New pairs of hands, or a new batch of enzymes, can be a problem.
Good Luck!
Jill
Dear Dr.Jill Miotke
Thanks for your valuable suggestion as i have mentioned the protocol that i am following from my previous time now a days i am following the same protocol but the attachment of cell to coverslip is low and i have changed everything like new media,Poly-D-Lysine and boric acid,Glutamex-I,B-27 supplement but not the L-Glutamic acid which i made around 3 month ago and the coverslip was purchased around 2 year before and before coating of coverslip with Poly-D-Lysine we usually keep it for 7 days in 60% nitric acid then wash brifly with Q water for 1 hr and dry it then coat with Poly-D-Lysine and keep it for 2 days and in medium containing well for another 2 days.
if you kindly give more specific suggestion it would be great for me
Dear Nazmul,
Since it sounds like you have a coating problem, you might also make new nitric acid, just in case it became contaminated and deposited something on the coverslips. When the poly-lysine is applied to the glass, if the glass is clean it should coat evenly.
Sometimes odd dirt and oil accumulates on glass--by any chance, were the coverslips stuck together in the box before you cleaned them? We sometimes get this film on our glass, even though it is stored in covered dishes in the tissue culture cabinet. The only way I have found to fix this is to soak them in 70% ethanol. If you are allowed to do this, try cleaning your coverslips in fresh 70% ethanol for about an hour (keep the container tightly closed!). This will also sterilize them if you then let them dry in the laminar flow hood. I hold each one individually with a pair of sterile forceps so that they dry one at a time, and when it is dry put it in the tissue culture well.
As a last resort, you might want to check your water quality. I once had a cartridge in my water purification setup go bad and leach something into the water. I only figured out something was wrong because my immunostaining no longer worked. I have access to supply rooms on our campus, so I quickly bought some sterile TC grade water. That fixed the problem.
How do you dissociate your cells before plating them? Any changes there?
Jill
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