Clonogenic assay with agressive cell line - (Jul/09/2009 )
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!
Maybe grow them in soft agarose matrix?
PurpleKnight on Jul 10 2009, 01:20 AM said:
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
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
