Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells
筆記録
– Sometimes, a stem or progenitor cell undergoes mutations, giving rise to a leukemic stem cell. In these cells, certain proteins are upregulated, and using anticancer compounds against such proteins can help control cells' growth. The effect of these anticancer compounds can be analyzed using CFU assay by monitoring the number and size of colonies formed.
Begin by taking progenitor cells from a leukemic mouse and diluting them in a suitable culture media. Add this cell suspension along with the appropriate anticancer compound to methylcellulose containing mouse-specific cytokines, and pour it into a Petri dish. Incubate this mixture till the colonies appear. Methylcellulose is a chemically inert, semi-solid matrix that prevents cell migration while cytokines stimulate progenitor cells to differentiate and form colonies containing recognizable progeny.
Next, stain the colonies using iodonitrotetrazolium chloride, or INT, and incubate overnight till the colonies turn dark reddish brown. Mitochondrial enzymes reduce yellow-colored INT to reddish brown-colored formazan dye. Finally, identify and count the colonies present on the plate. In the following protocol, we will perform a CFU assay to test the effect of anticancer compounds on YFP-positive cells.
– First, thaw the methylcellulose with cytokines for mice at room temperature. Then aliquot 3 milliliters of methylcellulose in a FACS tube. Add 100 microliters of the cell suspension with the appropriate inhibitors to 3 milliliters of methylcellulose. Then vortex the suspension on high for 10 to 30 seconds.
After most of the air bubbles escape, use a 1 milliliter syringe to plate 1 milliliter of the media in three replicate plates. Place the plates in a large Petri dish along with one open dish containing sterile water. Then cover the large Petri dish, and incubate the cells at 37 degrees Celsius.
Prepare iodonitrotetrazolium chloride stain by dissolving 10 milligrams of the iodonitrotetrazolium chloride in 10 milliliters of water. Filter sterilize the staining solution with a Luer-Lok syringe equipped with a 0.2 micrometer filter.
In the tissue culture hood, add 100 microliters of the staining solution dropwise around the CFU plate. Then incubate the plates overnight at 37 degrees Celsius in a cell culture incubator. Finally, after the colonies have turned a dark red-brown color, use a white background in the sterile tissue culture hood to take photos of the stained CFU plate.
