Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

1. Colony-forming Unit Assays

1. Thaw the methylcellulose with cytokines for mouse at room temperature for 1–2 h. Aliquot 3 mL of methylcellulose in a FACS tube using a 5 mL syringe without a needle. Sort the YFP-positive cells on a cell sorter.
2. Spin the cells down and suspend the pellet in IMDM complete media. Count the cells to be plated and dilute them to obtain 5,000 YFP-positive cells in 100 µL of IMDM complete media.
3. Add 100 µL of the cell suspension containing 5,000 YFP-positive cells and the appropriate inhibitors to 3 mL of methylcellulose. Vortex on high to mix it for 10–30 s.
4. After most of the air bubbles have escaped, use a 1 mL syringe to plate 1 mL of media in three replicate 3 cm plates by ejecting the media on one side and slowly tilting the plate in a circular motion to spread evenly.
5. The final concentration of the inhibitors to use are imatinib at 3 µM, DFC at 0.2 µM, and BCI at 0.5 µM, alone or in combinations. Wherever appropriate, delete c-Fos by plating the cells with 4-hydroxytamoxifen (1 µg/mL) added to the methylcellulose.
6. After plating, put the plates in a large Petri dish with one open dish containing 2 mL of sterile water in the middle. Cover the large Petri dish and carefully incubate at the 37 °C, 5% CO₂ incubator. Record the colony numbers after one week of plating by counting the total number of colonies under a microscope.
7. Analyze a few colonies from appropriate plates for c-Fos deletion by PCR. Collect the colonies by sucking them with a P1000 tip. Dislodge the cells in 500 µL of 1x PBS by washing the tip in the PBS multiple times. Spin the cell suspensions at 6,000 x g for 1 min and extract the DNA from the cell pellet using a genomic DNA isolation kit.
8. Use the genomic DNA for PCR using the primers mFos-FP3 and mFos-RP5. Analyze the PCR products on a 2% agarose gel and image using a gel documentation system.
9. For a graphic presentation of the colonies, stain the colonies, using Iodonitrotetrazolium chloride. Dissolve 10 mg of the Iodonitrotetrazolium chloride in 10 mL of water to obtain a 1 mg/mL solution. Filter sterilize the solution using a Luer lock with a 0.2 µm filter attached to a 10 mL syringe under sterile conditions in a tissue culture hood.
10. In the tissue culture hood, add 100 µL of staining solution dropwise around the CFU plate; be careful not to slide or disturb colonies. Incubate the plates overnight in a 37 °C, 5% CO₂ cell culture incubator. The colonies will turn dark reddish brown. Using a white background in the sterile tissue culture hood, take photos of the stained CFU plate.