Absorbance for MTT - What are the ranges of wavelengths for MTT detection and background? (Nov/25/2008 )

Hi all, I'm trying to set up MTT assays for cell proliferation, we bought the MTT from sigma aldrich (M2128) and the protocol states that we should measure formazan at 570nm and substract the background absorbance at 690nm.

The problem is the microplate reader that I am using only has absorbance filters for 560nm and for back ground, the only filter over 600nm is 720nm. Can I use 560nm for formazan and subtract absorbance at 720nm?

What does 720nm actually measure? please help!! thanks

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Hi
I do MTT without subtracting background. I have been using 540 nm for absorbance and also you can see many literature using the same at 570 nm. I dont think background influences so much as one can express % of proliferation/viability of control at the same condition.

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Thanks Thapa!

Right now I'm just collecting the readings of all available filters on the machine, so at least I can go back and retrieve the data if I need to without repeating everything again.

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The difficulty arises in not knowing the absorbance maxima of the MTT crystals generated. If you read at a different wavelength you may not be getting the full range of sensitivity out of the assay.

It also pays to know about the filters on your plate reader, are they narrow bandwith? long pass (and which direction?)? etc, all of which can have a bearing on the readings given.

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You're right bob1, I have to get the specs on the plate reader. Another thing, should I fix the gain on the reader for the wavelengths I am using or allow the reader to automatically adjust gain values each time I read a plate?

thanks heaps

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