mtt assay - problem in drug screening (May/19/2010 )

Hi there,
currently i am perforng mtt assay but i am facing problem in getting any consistent result in finding out ic50 value of the compound.i am getting same od for my blank and the wells with compounds.wavelength that i am using is 490 nm.please someone help me.

MTT is not measured at 490 - read the protocol! Measure at 570 with reference at 630.

Thanks for the reply. ya i know that but problem is that we do not have 570 nm wavelength (lack of facility) and i was also thinking may be because i am not using 570 nm that's why i am not getting result.but i did my optimisation at this wavelength and result was ok but when i started doing drug screening then problems started.

oasis on May 20 2010, 01:18 PM said:

even if u got any result i wud anyways never rely on that... the detection wavelength is different.. yiou cannot scientifically prove any argument right using this assay system!!!! look for a system were u can read at 570...!!!
moreover 490 adn 570 dont even fall in the same visible color spectrum range.... so its a big no no from my side!!!
sorry if this is not encouraging... but i wont rely on such data!!!

HEY thanks a lot for your reply, i will try with 570 nm .

if i go for 565 nm that will be ok or not??

oasis on May 20 2010, 03:19 PM said:

yeah tat is still much more better than your previous wavelength!!!! as the bandwidth of a 570 filter will anyways allow 565 nm light to pass!!!
Best luck!!! and as bob suggested it wud be good if u can have a reference wavelength at 630..

that's great i'll try this and let you know any change in my result. thanks a lot for suggestions to everyone.cheers !!!!

oasis on May 20 2010, 04:35 PM said:

Best luck oasis!!!

Yeah, Good luck, hopefully things work for you.

I am guessing measuring at 490 didn't work as it probably is not a part of the spectrum that is linearly related to the amount of formazan produced.