I am performing a soft agar colony assay using 2 different cell lines. Both are able to make colonies. So now I want to test a specific drug and see if it can affect their capability for making colonies. But I was wondering where should my drug be applied? Should I dilute my drug with the upper agar layer containing cells? Should I applied my drug above the upper layer? Or both? Thanks for your help.
Hi there
Both.
Concentration of your drug in upper layer will reduce over time due to diffusion. As I did not understand the meaning of "MY DRUG ABOVE THE UPPER LAYER", so what we do is to prepare the upper layer (soft agar) with drug,
1. Melt the soft agar and cool it upto tolerable range.
2. Mixed the required amount of drug.
3. Mix the cell.
4. Pour the mixture above the first layer with the drug.
Hi,
For Ames test, I always used soft agar for homogeneously mixing the test drugs with cells. You can preincubate your drugs and cells in this soft agar too. When you mix the cells with a drug /compound in a certain volume, you can accelerate interaction between the drug and cells.
good luck
Hi there,
The only way to get sure about the actual drug concentration in the immediate cell environment is to add it to the soft agar... If you add the drug at the surface you will have no clue about local concentration.
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