Sean Lauber:MTT Cell Proliferation Assay: Difference between revisions
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5. Incubate the cells at 24-72 hours at 37°C/5% CO2. | 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. | 6. Add 20 ul of 5 mg/ml MTT (ensure that the MTT is warmed and completely dissolved) directly to the well. If you want to recover the supernatant, you can do so, but be sure to add another 100 ul of PBS or similar to make the final volume 120 ul. | ||
7. Incubate for 4 hours at 37°C in the dark. | 7. Incubate for 4 hours at 37°C in the dark. | ||
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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). | 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). However this is unnecessary since you're probably going to be expressing it as a % growth from untreated cells (untreated cells = 100%) | ||
In one situation I had too many cells and the absorbance was out of the detectable range. In this case you can dilute the solution with some amount of PBS and re-read the absorbances (1:2, 1:4, etc until it's on scale). Be sure to dilute all samples if you want to be able to compare them. | |||
MTT solvent: | MTT solvent: | ||
8 mM HCl | 8 mM HCl | ||
0.2% IGEPAL CA-630 (in isopropanol) | 0.2% IGEPAL CA-630 (in isopropanol) | ||
Revision as of 21:29, 10 July 2013
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. Grow LLC cells up and split after 48 h
2. Seed 2000 cells onto a 96-well plate (in 2% FBS) and allow the cells to adhere to the plate for 2 hours
3. Replace with media containing whatever you're studying with 2% FBS(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. If you want to recover the supernatant, you can do so, but be sure to add another 100 ul of PBS or similar to make the final volume 120 ul.
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). However this is unnecessary since you're probably going to be expressing it as a % growth from untreated cells (untreated cells = 100%)
In one situation I had too many cells and the absorbance was out of the detectable range. In this case you can dilute the solution with some amount of PBS and re-read the absorbances (1:2, 1:4, etc until it's on scale). Be sure to dilute all samples if you want to be able to compare them.
MTT solvent: 8 mM HCl 0.2% IGEPAL CA-630 (in isopropanol)
