Basically, I make dissociated neuron cultures grown on glass coverslips coated with poly-L-lysine. While the density of neurons tends to be quite high on the edge of the coverslip it is much lower in the center...Is there a way I can fix that issue?
This is my current protocol: I prepare a solution of poly-L-lysine hydrobromide dissolved in pH8.5 borate buffer at a concentration of 0.1mg/ml. I put a 200 microL drop of that solution on each 13mm coverslip, leave in the incubator overnight and wash three times using distilled water before adding my culture medium to the dish.
Thank you.
Hello Cécile,
I use to pre-treat glass coverslip with 1mg/ml of poly-L-lysine, making 4 small drops at the four extremities of each coverslip (a total amount of 130uL are usually enough for 13mm and 150 for the 25mm ).
The distribution of your culture may depends from the technique you use to plate your neurons. In our lab we use to plate the needed amount of cells in the same volume and with the same technique used for the pre-treatment with poly-L-lysine
Of course when you plate your cells try to avoid any formation of bubbles in the center of the coverslip.
Let me know if it works!
All the best
Davide
Hello Cécile,
I use to pre-treat glass coverslip with 1mg/ml of poly-L-lysine, making 4 small drops at the four extremities of each coverslip (a total amount of 130uL are usually enough for 13mm and 150 for the 25mm ).
The distribution of your culture may depends from the technique you use to plate your neurons. In our lab we use to plate the needed amount of cells in the same volume and with the same technique used for the pre-treatment with poly-L-lysine
Of course when you plate your cells try to avoid any formation of bubbles in the center of the coverslip.
Let me know if it works!
All the best
Davide
Hello Ceclie,
I agree with Davide that it is important to be careful with your plating technique. Bubbles are bad, and are sometimes tough to avoid.
However, coating coverslips is notoriously tricky, since it can depend on how clean they are to begin with, and not all suppliers are good at cleaning them. We would also autoclave them in our own distilled water autoclave, since industiral and academic autoclaves can have contaminants in their steam that would lead to uneven coating. What does the poly-lysine drop look like on the coverslip? Does it sheet nicely across the surface?
Another problem may be that the poly-lysing is drying out a bit in the incubator overnight, so that there is a higher concentration at the edge of the coverslip rather than in the center. In fact, that is how some people used to creat protein gradients for their growth cone pathfinding studies, although with much smaller volumes. It can be dificult to detect such drying, so I used to make sure that my culture dishes with coverslips were kept in a secondary humidified chamber. Since southern California can get pretty dry, sometimes it was easier to just bit the bullet and put 1 ml of poly-lysine in the 35mm culture dish, rather than try to just coat the coverslip. If you decide to do that, increase the number of washes with water to 5 or 6, since more poly-lysine gets trapped under the coverslip, and excess poly-L-lysine can be toxic to cells. (For that reason, I have always used poly-D-lysine, to be on the safe side.)
I solved most of these problems by switching to glass-bottomed culture dishes, since the techniques I use require an inverted microscope.
Good luck!
Jill
I might perhaps have not got your question correct, Cecile, but i have a very basic doubt from what i understand. One, is your initial seeding density optimal (not too low/ or too high)? and second, the initial amount of media you take to plate the cells on your coverslips. Generally, many researchers initially use small amount of media with optimal density, to cover the specific area( the center of the coverslip in your case), incubate for sometime (half an hour or so) for the cells to attach mostly on the required region and then make the final desired volume. Again, of course Proper PLL coating is a requisite, but you might want to check if this works for you. it sure works for me. Hope it helps.
Thanks for your helpful advice about the coating of the coverslips. Thank you also for mentioning the seeding of the cells in your comments (see Davide's and Poojashri's answers); this could indeed contribute to the problem as I usually add my cells to the culture dish filled with growing medium rather than 'dropwise' on the coverslips.
Hello Cecile,
I would like to recommend an easy solution for this. While seeding a particular number of cells on coverslip, just take the entire number of cells in a small volume of FBS or any viscous liquid which is compatible with cells and place it in center of coverslip and let it be undisturbed say for 5-10 minutes. Once neurons are adhered there then you can add rest of your solution and incubate the same accordingly.
Hope it helps !
I find that the slide chamber is the best, no coverslip anymore, and you can stain, wash with ease. Check http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_646413__-1_0
I had exactly the same issue when seeded the cells on cover slips, no matter coated or non-coated surface. Finally, right after seeding cells in the wells, I shake the plates a few times clock-wise or anti clock-wise very slowly. The cell density looks better in the center of the cover slips. Hope this helps. Good luck!
Thank you Li. Actually, as suggested by few people here, I tried plating the cells using the 'spotting technique' (see Jakub's comment) and it made a huge difference. The culture looks more even and the density per coverslip is much higher.
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