Wednesday, May 04, 2005

Description
Allows one to test the capability of adherent cells to survive and replicate following insult with chemicals or radiation.

Procedure
1. Grow the cells in regular media to 70% confluence and treat the cells with the appropriate chemical or radiation dose. Be sure to include a non treated control.

2. At the required endpoint, trypsinise and count the cells as usual.

3. Re-seed 1000 cells into a new 60mm or 100mm tissue culture dish (in duplicate) and incubate for 9 days. Fresh media should be added at day 5. (See Tip #1)

4. At day 9 (or when the cell colonies have grown to approx 50 cells) remove the media and add 1ml (for 60mm dish) or 2ml (for 100mm dish) of Clonogenic Reagent. Leave at room temperature for 45 minutes.

5. Wash the cells twice with PBS and count the blue colonies.

6. The data can then be expressed as percent survival relative to the control: ((average treated count)/(average ctrl count))x100

Recipes
CLONOGENIC REAGENT
50% Ethanol
0.25% 1,9-dimethyl-methylene blue

Supplies
1,9-dimethyl-methylene blue from Sigma Aldrich (Cat.# 34,108-8)

Tips
Tip #1: Seeded at such a low density some cells have difficulty adhering to the plates. It may help to use Cell Plus plates from Sarstedt to increase adherence OR poly lysine treat the plates.