Hi,
I am trying to perform a clonogenic assay using HCT116 cells, but for some reason, the plating efficiency of control is very low. The cells do not seem to form colonies. In our lab we conduct a clonogenic assay with success using other cancer cell lines. Since the cells are plated on poly-L-lysine coated dishes, I suppose that it could be a disturbing factor. Does anyone has an idea what might be a problem? We seed 500 cells/60 mm dishes in 6 ml of culture medium and incubate in 37degrees for 14 days.
Thanks in advance.
Hi,
I don't think that it is the poly-L-lysine coat is causing the problem. I believe it is the overall surface area of the 60mm dish causing the problem.
I would suggest scaling down the size of the dish either by using a 6-well plate or a 35mm dish instead.
Another modification that you can add to your protocol would be standardize the minimum amount of cells required. Keep different plates with varying cell count like 500, 750, and 1000 cells/plate. This would give you an idea as to what should be the optimum cell number for them to start forming colonies.
Every cell line has its own characteristic and their proliferation and ability to form a colony would hence vary. But yes, I certainly believe that scaling down the size of your culture dish and optimizing the correct cell count would solve your problem. It works well for us.
Feel free to ask if you have any doubts.
Hope this helps.
Hi we usually plate 1000-2000 cells in 6 well plates.
The critical step is to not plate your cells from a confluent plate but from a plate 70% confluent (that have not been over confluent the previous passage too)
Hope it help
The problem is solved. Indeed, I should increase the number of cells. Now I plate 1000 cells/60 mm dish.
Thanks for help.
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