MTT Assay troubleshooting - (Aug/21/2008 )

We are having issues with the consistancy of our MTT assay from the ATCC. We are using the test to measure cell counts off a sample surface. All the reagent volumes are scaled up to meet the volume requirements of a sample in a 12 well plate.

The issue we are encountering is our error bars on both control and treated samples are massivly high. Assuming that there is nothing wrong with the method, what might be possible sources of error.

Initial seeding = 1000 cells/cm^2

Also, is there an alternate testing method other than MTT or standard dye counting by hand that others have had success and consistent results with?

Thanks in advance.

My lab uses the MTT assay with no issues - but then again, we seed 500 or 5000 cells/well in a 96-well plate, which seems to be a higher density than yours, so you may want to up the cell concentration. Did you do the cell dilution test they recommend in the kit instructions?

As for other sources, there's also the MTS assay, which uses a slightly different reagent but also makes formazan crystals. Then there's the tritium incorporation assays, where you let the cells grow in the presence of radiolabeled nucliotides and then quantify the signal you get from the samples, but why bother with radioactive stuff when colorimetric assays are available.

My lab switched to WST (water-soluble tetrazolium) last year which is another derivate of MTT.
we found that it yielded more repeatable results than the MTT assay because you skip the adding of isopropanol.

when you say error bars is it the well-to-well variance, plate-to-plate variance or the variance between several measurements of the same well? all of these errors require different kinds of troubleshooting.

Solubilization method? I got more consistency after switching from acidified ethanol to 1% SDS overnight solubilization.