Sean Lauber:MTT Cell Proliferation Assay

The following protocol has been optimized for growing lewis lung carcinoma (LLC) cells over a 2 day period. However I have been able to grow the cells over a 3 day period but this was less consistent.

1. Seed 2000 cells onto a 96-well plate to allow the cells to adhere to the plate.

2. Incubate overnight at 37°C/5% CO2.

3. The following morning, replace the media with 2% FBS containing media if required.

4. Add cytokines or whatever you're studying (final volume = 100 ul)

5. Incubate the cells at 24-72 hours at 37°C/5% CO2.

6. Add 20 ul of 5 mg/ml MTT (ensure that the MTT is warmed and completely dissolved) directly to the well. DO NOT REMOVE the media from the well to store for analysis. LLC cells are a mixed adherent/suspension population.

7. Incubate for 4 hours at 37°C in the dark.

8. Add 120 ul of the MTT solvent solution (warmed).

9. Pipette up and down vigorously to lyse the cells and dissolve the colorized product. If you look at the bottom of the plate and notice specks of purple, then you haven't pippeted well enough. Everything should dissolve. If it doesn't dissolve, put it on a shaker for 15 minutes (in the dark), and then try again.

10. Read at 550 nm.

You might find it useful to include a standard curve. For this I would plate 500, 1000, 5000, 10000, 30000, 70000, 100000 cells shortly before treating with MTT (allowing time for adherence).

Normally I prepare this data as compared to the growth of the control (=1.0).

 MTT solvent:
 8 mM HCl
 0.2% IGEPAL CA-630 (in isopropanol)