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trouble to grow cells on top of coverslip


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#1 minylim

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Posted 11 September 2009 - 12:40 PM

I've been trying to grow adenocarcinoma cells on top of coverslip (HCl washed) but I found more cells grew at the bottom of coverslip (space between coverslip and 12-well plate).
I used chamber slides but couldn't get the same result when I prep cells on 12 well plate....agrrrrr
Should I glue coverslip with vaseline or whatever? :P Please help!!!!!!!!!

#2 mikew

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Posted 11 September 2009 - 01:25 PM

Some cells do not grow well on cover slips unless they are poly-lysine coated.
However, I normally submit the cover slips to UV light in the hood to sterilize and then seed.
I don't think you need to HCL wash the slips, this may change the charge of the cover lsips and lead to less binding.
I put the slip into a 24 well plate add media and THEN add the cells. This order seems to work well.
Also, using fresh media makes a difference.

#3 Alfred Nobel

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Posted 11 September 2009 - 01:49 PM

Try putting the cells plus about 300-500 ul of buffer on the top of your slides only. Let the cells attach in the incubator for about an hour or two, then add additional buffer to flood the well. As to sterilizing, I've been dipping the cover slips in EtOH then letting them dry under UV light which has been working fine for cancer cells.

#4 minylim

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Posted 11 September 2009 - 02:12 PM

It's epithelial cells that adhere very well to any surface, I don't think poly lysin coating is required. HCl washing is mainly to clean the surface and to improve cell binding by generating Si-O (I heard so). I autoclaved them in ddH20 so it must be clean enough. =)

Solution will be how to prevent cells get in between coverslip and culture dish. I'll try the not-to-spill-over method, wish me good luck! (I have coworkers tend to slam the incubater door...) Thank you!!!

#5 Stephan

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Posted 14 September 2009 - 02:02 AM

I agree with Alfred Nobel. Seed your cells in a small amount of media, not more than would cause the media "pool" to run off of the slide (balance is of great importance here). Once the cells have adhered you can fill with more media. You could even leave them overnight to attach - the amount of media in the pool should be enough for such few cells.
As for the sterilizing - I use 70% ethanol and leave to dry. Never had a problem before.

#6 Bill Nye

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Posted 15 September 2009 - 06:32 PM

I coat my (glass) coverslips with 2% gelatin and they adhere very well. You should try preparing your coverslip-dishes ahead of time. I use 22x22mm coverslips inside 35mm dishes. Try this:

- use the lid of a 35mm dish to hold about 1ml of 2% gelatin solution
- take your autoclaved coverslips and dip them into the gelatin solution (do both sides)
- take the coated coverslip and place it inside another 35mm dish
- close the dish and let the gelatin dry (I usually turn on the UV and let it dry overnight)
- repeat the above steps to make as many dishes as you need (I usually make stocks)

In this method, the coverslip is adhered to the bottom when you initially seed the cells, so this prevents the majority of cells from sneaking to the bottom. Don't worry about the coverslip being stuck, once the coverslip is submerged in media, it will eventually be "unstuck".

Edited by Bill Nye, 15 September 2009 - 06:34 PM.


#7 minylim

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Posted 16 September 2009 - 09:24 AM

Thank you Bill! I'll try your coating method as well. I guess I can use other matrix than gelatin...not sure I have it in lab... =)




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