When you do soft agar colony formation assay, do you treat (with drug/ modulator) cells growing on agar or you pre-treat the cells for a particular incubation period and then seed the treated cells on agar for the colony-formation assay (for 21 days)?
It would depend on the question you are asking: whether a drug has an effect on cell proliferation, survival, or colony formation. If the later, the drug needs to be present in the soft agar during the incubation period. If the drug is known to have a short half-life it could be challenging to maintain a desired drug concentration during the assay period.
@Divaker Choubey, would repetitive dose of drug (say once in every three days) help during the 21 day incubation? If i want to check the effect of protein overexpression on colony formation, should I transfect the expression plasmid every 3-5 days in soft agar cells?
Would it be at all possible to transfect the cells in soft agar?
then you can try with normal colonogenic assay, as what i have done. Plate ~500 cells in 12/6 well plate (depending on doubling time) incubate with the drug (at IC50 and IC90) for specified period, then change the media and let it grow for in between 7 to 15 days and then do crystal violet staining.
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