- The colony formation assay is an in vitro analysis of an anticancer drug on the cancer cell's growth, proliferating and differentiating into colonies in semi-solid media. To begin, seed cancer cells in the RPMI medium with growth supplement-- fetal bovine serum, or FBS, and antibiotic. Incubate the cells for the required duration.
Next, collect the desired concentration of cells in a microfuge tube and centrifuge. Re-suspend the pellet in PBS. Prepare methylcellulose-based medium, and dispense it to different tubes with FBS to promote optimal growth.
Add an appropriate amount of cell suspension in tubes, and vortex them to mix the cells with the medium. Next, add anticancer compounds in tubes, and prepare a control by adding DMSO. Vortex tubes.
Add this cell mixture to the culture plate, and incubate for the desired duration. After incubation, add methylthiazol diphenyltetrazolium bromide, or MTT, and incubate for some time. MTT reduces yellow tetrazolium salt in viable cells to purple formazan crystals. Viable colonies turn violet. Capture the images using a digital camera, and analyze the viable colonies. In the following protocol, we will study colony formation assay for assessing the activity of hydroxycoumarin OT48 and BH3 mimetics in lung cancer.
- To begin, plate 20,000 A549 cells per square centimeter in 15 milliliters of RPMI 1640 cell culture medium, supplemented with 10% FBS and 1% antibiotics in a 75-square-centimeter flask. Incubate the culture at 37 degrees Celsius and 5% CO2. On the day of the experiment, use a hemocytometer to determine the cell concentration.
Collect 50,000 cells in a 1.5-milliliter microcentrifuge tube by centrifugation at 400 G for seven minutes. And re-suspend the cells in 100 microliters of sterile 1X PBS. Using a multi-pipette, dispense 1.1 milliliters of semi-solid methylcellulose-based medium, or MCBM, into tubes, preparing one tube for each condition. Then add 110 microliters of FBS to each tube.
Next, seed 1,000 cells per milliliter. Then, pipette 2.4 microliters of the stock cell solution into the tubes of MCBM and FBS. After adding the cells, hold the tubes vertically and vortex them at the highest speed for one minute to thoroughly mix the MCBM and cells.
Now, add test compound OT48 alone, or in combination with A1210477, to the samples. Prepare a separate tube as a control for the solvent used, such as DMSO. Then vortex the samples for one minute.
For each assay, pipette one milliliter of the mixture into the center well of a 12-well cell culture plate. Then use one milliliter of sterile water or PBS to fill the empty wells. Incubate the plates at 37 degrees Celsius and 5% CO2 for 10 days. Then add 200 microliters of a five-milligram-per-milliliter MTT stock solution to each well. After incubating the plates for 10 minutes, check for viable colonies, which will turn violet.