I have tried clonogenic assay for Sk Hep1 cell line 4-5 times. But the clonogenic efficiency in control in itself is less than 10%. I have tried seeding up to 1000-3000 cells in 6 cm as well as 3.5 cm plate without any success. due to such low colony-forming efficiency, I cannot proceed with the experiment. Can anyone suggest a possible reason for it &/or solution?
I have no experience in dealing with SK-HEP-1 cells, but plating efficiency of a bit less than 10% is not too low for colony formation assay. The increased number of plated cells, the increased FBS concentration, pretreatment with a coating agent (e.g., poly-L-lycine, fibronectin, collagen, gelatin) may help.
Hi Megha Tawate
I usually perform my assays in triplicate in a 6-well plate. I use 0.6% agarose as a base layer and seed 20,000 cells/well in 0.3% agarose. Many cells types struggle to survive in these conditions so you may also resuspend your cells in conditioned media to aid viability.
Good luck.
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