Problems with MTT Assay and color changes - MTT Assay and Cobalt Chloride (Aug/10/2006 )

Good Afternoon all,

I have recently started working in a new lab with human glioma cells. I have done the MTT assay several times in my old lab. However, I treated the glioma cells with cobalt chloride and then added MTT (0.5mg/ml) for 3.5hrs and solubolized in DMSO.The control cultures turned dark purple (which I am use to seeing) however the cobalt chloride treated cultures turned blue and the intensity of the blue increased with an increase in cobalt chloride concentration. I have never seen this before, therefore the MTT assay is telling me that an increase in cobalt chloride increases cell viability which should not be true. I have also washed the cells with PBS before adding DMSO and the color is not as prevelant but the results remain the same. Has anyone ever experienced this problem with MTT and cobalt chloride? ANY suggestions would help.

There seems to be a complex formed between the two. Never done anything like this before, but it maybe worth trying using 2x as much of EDTA to pull off Co from the complex.

I don"t know, maybe I 'll realise in few minutes that it's stupid.

but may be you could add the same amount of cobalt chloride to all cells after DMSO, or just in the same time? So you would be able to compare again.
But I don't know if the change of color is still proportional to cell death? you would maybe have to make a kind of standard curve?