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Clonogenic assay with agressive cell line


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#1 PurpleKnight

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Posted 09 July 2009 - 03:20 PM

Hi everyone,

I am doing clonogenic assays to measure the sensitivity of my cell line of interest to ionizing radiation. I first performed a preliminary experiment with mock treatment and seeded my cells at different densities in 6 wells plate to know what to expect in term of number of colonies and the incubation time needed to get colonies of 50 or more cells.
The problem I am facing is the following: the cells don't form nice isolated colonies, whatever the density they are seeded at. It seems that the daugther cells are migrating away from the mother cells after division leading to a "colony" that looks widespread instead of compact and well delineated, and therefore not as easy to count.
So I am wondering what to try next to improve this. Coating the plate with poly lysine? As any of you faced the same kind of issues and most importantly how did you overcome them!
Thanks a lot in advance!

#2 genehunter

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Posted 10 July 2009 - 06:45 AM

Maybe grow them in soft agarose matrix?

#3 klinmed

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Posted 10 July 2009 - 07:03 AM

Hi everyone,

I am doing clonogenic assays to measure the sensitivity of my cell line of interest to ionizing radiation. I first performed a preliminary experiment with mock treatment and seeded my cells at different densities in 6 wells plate to know what to expect in term of number of colonies and the incubation time needed to get colonies of 50 or more cells.
The problem I am facing is the following: the cells don't form nice isolated colonies, whatever the density they are seeded at. It seems that the daugther cells are migrating away from the mother cells after division leading to a "colony" that looks widespread instead of compact and well delineated, and therefore not as easy to count.
So I am wondering what to try next to improve this. Coating the plate with poly lysine? As any of you faced the same kind of issues and most importantly how did you overcome them!
Thanks a lot in advance!


Try plating your cells exactly as you are doing.
1, Allow them to adhere.
2, Remove the medium
3. Gently overlay with complete tissue culture medium plus 0.5 % low gelling temp agarose (eg Seaplaque. 2ml/ well). This can be made by adding 1 ml of autoclaved 5% agarose (use a syringe to handle) to 9 ml medium (37oC).
4. Allow to set at RT and incubate

5. After the incubation you can stain the colonies by overlaying the agarose with 0.01% neutral red (ca 1ml) for 1 h. Then aspirate stain, incubate for 1 - 2 hours and then count. Alternatively you can use MTT.

Hope this helps

#4 PurpleKnight

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Posted 10 July 2009 - 10:34 AM

Try plating your cells exactly as you are doing.
1, Allow them to adhere.
2, Remove the medium
3. Gently overlay with complete tissue culture medium plus 0.5 % low gelling temp agarose (eg Seaplaque. 2ml/ well). This can be made by adding 1 ml of autoclaved 5% agarose (use a syringe to handle) to 9 ml medium (37oC).
4. Allow to set at RT and incubate

5. After the incubation you can stain the colonies by overlaying the agarose with 0.01% neutral red (ca 1ml) for 1 h. Then aspirate stain, incubate for 1 - 2 hours and then count. Alternatively you can use MTT.

Hope this helps
[/quote]

Thanks for your advices, I like that idea and will definitely try it! Like I said in my previous post, I just want to look at the cells ability to form colonies after treatment (and not perform an anchorage-independent growth assay) so this strategy should work to that respect! Do you think crystal violet stain would work as well as the neutral red? I guess this will be the next thing for me to test!
Thanks again for your help!
B)

#5 PurpleKnight

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Posted 10 July 2009 - 10:37 AM

Maybe grow them in soft agarose matrix?


Thanks for your suggestion!
I will try to overlay my plates with some sort of soft agarose matrix once my cells have attached to the plate!




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