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

Samenvatting

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).

This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

Protocol

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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
IMDM	Cellgro (corning)	15-016-CVR
MethoCult GF M3434 (Methylcellulose for Mouse CFU)	Stem Cell	3434
4-Hydroxytamoxifen	Sigma	H6278
Recombinant Murine SCF	Prospec	CYT-275
Recombinant Murine Flt3-Ligand	Prospec	CYT-340
Recombinant Murine IL-6	Prospec	CYT-350
Recombinant Murine IL-7	Peprotech	217-17
DFC	LKT Laboratories Inc.	D3420
BCI	Chemzon Scientific	NZ-06-195
Imatinib	LC Laborator	I-5508
1x PBS	Cellgro (corning)	21-040-CV
1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2	Becton Dickinson	329461
Iodonitrotetrazolium chloride	Sigma	I10406
Instruments
NAPCO series 8000 WJ CO2 incubator	Thermo scientific
Swing bucket rotor centrifuge 5810R	Eppendorf
C-1000 Thermal cycler	Bio-RAD
ChemiDoc Imaging System (imaging system for gels and western blots)	Bio-RAD	17001401
Mice
ROSACreERT2	Jackson Laboratory
ROSACreERT2/c-Fos fl/fl Dusp1-/	Made in house
ROSACreERT2/c-Fosfl/fl	Made in house