Colony Formation Assay: Assessing the Efficacy of Anticancer Agents on Colony-Forming Lung Cancer Cells

Protocol

1. Colony Formation Assays

1. Culture A549 cells at a concentration of 20,000 cells/cm² in 15 mL of RPMI1640 cell culture medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics in a 75 cm² flask in a CO₂ incubator at 37 °C and 5% CO₂.
2. On the day of the experiment, determine the cell concentration by using a hemocytometer. Collect 50,000 cells by centrifugation at 400 x g for 7 min in a 1.5 mL microcentrifuge tube and re-suspend cells in 100 µL of 1x sterile PBS (Phosphate Buffered Saline) in 1.5 mL tubes.
3. Dispense 1.1 mL of semi-solid methylcellulose-based medium (MCBM) in 15 mL tubes with a multipipette. Prepare one tube for each condition.
4. Add 110 µL of FBS to 1.1 mL of MCBM using a P200 micropipette.
5. Seeds cells at 1,000 cells/mL. Collect 2.4 µL of cell suspension from the stock solution (50,000 cells in 100 µL of 1x PBS) using a P2 micropipette and add to 1.1 mL of MCBM supplemented with 10% FBS.
6. After adding the cells, vortex the tubes for 1 min, holding them vertically at the highest speed to mix MCBM and cells well.
7. Add test compounds (OT48 alone or in combination with A1210477). Prepare a separate tube as a control for the solvent used (here dimethyl sulfoxide (DMSO)). Depending on the concentration of the compounds and the volume added for each condition, the solvent control will include the highest concentration of solvent used for the dilutions of the test compound, to assess for adverse effects caused by the solvent.
8. Vortex tubes for 1 minute.
9. For each assay, add 1 mL of the mixture with a P1000 micropipette to the center well of a 12-well cell culture plate.
10. Fill empty wells with 1 mL of sterile water or 1x PBS to stabilize humidity.
11. Incubate culture plates for 10 days in an incubator at 37 °C and 5% CO₂
12. After 10 days of incubation, add 200 µL of an MTT (methylthiazolyldiphenyl-tetrazolium bromide) stock solution (5 mg/mL) to each well to reach a final concentration of 1 mg/mL.
13. Incubate 10 min in an incubator at 37 °C and 5% CO₂. Viable colonies will turn violet.

Materials

Name	Company	Catalog Number	Comments
A549	ATCC	CCL-185	37 C°
RPMI 1640	Lonza	30096	4 C°
FBS	Biowest	S1520-500	-20 C°
Penicillin-Streptomycin	Lonza	17-602E	-20 C°
Cell culture flask T75	SPL	70075	RT
PBS solution	Hyclone	SH30256.02	RT
1.5ml tube	Extragene	Tube-170-C	RT
15 ml tube	Hyundai Micro	H20015	RT
12 well plate	SPL	30012	RT
MethoCult	StemCell technologies	4230	-20 C°
Thiazolyl Blue Tetrazolium Bromide (MTT powder)	Sigma	M5622	4 C°
LAS4000	GE Healthcare Technologies		RT