Count cells from both wild-type and mutant cell lines using a counting chamber or automated system. Prepare a cell suspension with a defined concentration of 1000 cells per milliliter by diluting the appropriate volume of cell suspension in culture medium.
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2
Seed cells into culture plates
Pipette 1 ml of the prepared cell suspension into each well of a 6-well plate to achieve equal cell density of 1000 cells per well. Allow cells to attach to the plate overnight.
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3
Treat cells with drug concentrations
The following day, treat cells with increasing concentrations of the drug of interest, with two untreated control wells per cell line. Prepare technical duplicates for each treatment condition.
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4
Incubate plates for colony formation
Incubate the seeded and treated plates for 7 to 14 days depending on cell line and compound, allowing single cells to proliferate into visible colonies.
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5
Fix and stain colonies
Remove the medium and fix cells in 70% ethanol for 10 minutes. Allow plates to dry, then stain with crystal violet dye for several minutes to visualize colonies.
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6
Wash plates and count colonies
Wash away the background stain with water so only the stained colonies remain clearly visible. Count the colonies in all technical duplicates and calculate averages.
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7
Analyze and plot results
Set the untreated control to 100% and normalize all other conditions relative to the control. Plot the data to compare drug susceptibility between cell lines and determine treatment effects on cell survival.