Noble Agar Assay

Description
Cancer cells do not show anchorage and contact inhibition of growth. To assess the anchorage and contact independent growth of cells, noble agar assay can be utilized. Noble agar is used to coat the tissue culture plates to cover the polylysine coating of the plate. On this noble agar coated, anchorage independent tissue culture plates normal and cancer cells will be seeded in very low density to prevent the contact between cells. Plating efficiency is a measure of the clonogenic ability of cells and Cancer cells will have higher plating efficiencies than normal cells.

Procedure
1. Make 1.2% and 0.6% noble agar medium in dw.
2. Autoclave both noble agar medium amd equilibrate at 42 degree celsius.
3. Prepare 20% FBS containing DMEM/RIPA or any other tissue culture medium according to your cells. These tissue culture medium should not contain phenol red.
4.Also equilibrate the tissue culture medium at 42 degree celsius.
5. Mix the 1.2% noble agar and 20% DMEM medium and precoat the 6mm polystyrene flat bottom of tissue culture plates with 0.6% noble agar in DMEM supplemented with 10% FBS.
6. Allow it to solidify for 5-7 min.
7. Mix the 0.6% noble agar and 20% DMEM medium to make 0.3% noble agar in DMEM supplemented with 10% FBS.
8. Harvest cells from tissue culture flasks by Trypsin/EDTA.
9. Wash the cells with PBS.
10. Resuspend the cells in 0.3% noble agar in DMEM medium containing 10% FBS at a density of 1500 cells/ml.
11. Plate 4500 cells on 0.6% coated tissue culture plates by pouring the 3 ml of cells in 0.3% noble agar in DMEM medium containing 10% FBS.
12. Allow this to solidify for 5-7 min. and then cover it with 20% FBS containing DMEM tissue culture medium.
13. Incubate the plates at 37 degree celsius for 2-3 weeks.
14. Change the 20% FBS containing DMEM medium twice a week.
15. Number of clones will be counted by phase contrast microscopy after 14-21 days.