MTT: absorbance over the control - (Apr/15/2008 )
Hi
I have a problem with MTT test. I am coming to the conclusion this assay is inadequate for check cells proliferation.
I grow T98G cells in standard medium and medium with tested compounds (antagonists for GPCR inhibiting mitotic activity).
Using hemocytometer I confirmed many times that my drugs decrease cells proliferation.
But when I conduct MTT test, I observe reverse resutls. I seed cells in the same number per each well and gain the highest absorbance in the highest used concentration of my drug in comparision to control! Control absorbance is always the lowest! How can I interptrete these data? In my opinion MTT test measures metabolism level, not number of cells! Is it possible that my chemicals induce higher mitochondrial dehydrogenase activity, that results in increased amount of reaction product (purple formazan), although I am sure that there are less cells than in control?
Help!
Neuronella
Hi there,
I have been doing MTT for the past year!! And I am studying proliferation for my drugs to the GPCR as well!!
May be you can try doing MTT at time point intervals. that way you can see the rate of growth for ur partucular type of cells. What u can also do wud be an optimal cell count (example, seed 1000, 2000, 3000, etc etc in a 96 well plate and do the MTT the next day, this way u wud know how many cells are there in ur plate after n number days while u do ur expt)...
if u can be a bit clear on what kind of chemical u r uing then we can try to work out the possiblity of ur chemiacal inducing mitochondrail dehydrogenase activity!!
But other than this I shall think about it and let you know.. You can also count the deal cell : live cells ratio in ur sample....
Good luck with it...
I have a problem with MTT test. I am coming to the conclusion this assay is inadequate for check cells proliferation.
I grow T98G cells in standard medium and medium with tested compounds (antagonists for GPCR inhibiting mitotic activity).
Using hemocytometer I confirmed many times that my drugs decrease cells proliferation.
But when I conduct MTT test, I observe reverse resutls. I seed cells in the same number per each well and gain the highest absorbance in the highest used concentration of my drug in comparision to control! Control absorbance is always the lowest! How can I interptrete these data? In my opinion MTT test measures metabolism level, not number of cells! Is it possible that my chemicals induce higher mitochondrial dehydrogenase activity, that results in increased amount of reaction product (purple formazan), although I am sure that there are less cells than in control?
Help!
Neuronella
Hi
I am very gratefull for your explanation, surely I'll try to take into account your advice, but now I have no time to repeat my experiment (I'm preparing poster for the conference so I needed the interpretation for my results).
My drugs are peptides, that sequences are mixed: each compound is a peptidic combination of substance P antagonist and opioid receptor (MOR or KOR) agonist. Now, when you find out what type of my drugs are, can you confirm or exclude my hypothesis concerning methabolism?
Thank you for help
Neuronella