solvent for formazan crystals in MTT assay - (Oct/04/2006 )

Hai,

I have been working with MTT assay and I was using DMSO to diisolve the formazan crystals. Now I am using isopropyl alcohol as there is a shortage of DMSO in our lab.So now i have doubt in the reproducibilty and stability of reading when compared to DMSO.Is isopropyl alcohol is a good and recommended solvent to dissolve formazan crystals.Is it affects the stability of formazan crystals as formazan crystals itself is very unstable one.Can anyone help me knowing that

Thank you

Hi,

I used acidified detergent (10 to 20% SDS in 0.4 to 0.5N HCl) to dissolve formazan crystal w/o problem.

Caveat though, don't store it more than neccessary as SDS and HCl will form insolubles after more than 2 days. Best is to read it after o/n or > 6 hours incubation at RT in the dark. Oh ya, don't pipet up and down to dissolve the formazan as there's SDS. Mix gently and leave it.

Bests

Yes, 20% SDS in 0.5N HCL works pretty well. But as said in previous post, beacause mixing with pipette forms bubbles, don't mix it using pipette. Best thing is to leave it overnight and read the plate next day morning

hi all,
i'm slightly off the track, but wanted to know - whn working with suspension cells & MTT, is it important to remove growth or drug containing media, prior to the addition of MTT containing one. If yes , is it by centrifugation?
I m using MTT for concentration dependent effect of a drug, but not getting sharp gradation in that term.
kindly help.

Hi Sandhya,

Using round bottom plates for suspension cells, helps in centrifugation of formazon crystals. You can just remove the supernatant leaving behind formazan crystals and then add solubilization solution to the wells. But, I have had variations while using suspension cells for MTT assay.

Hai,

I think your query is to know whether you should remove the media containing drug before MTT addition?

in that case no need to remove the media , you can add MTT solution to the wells containing media , and see to it that each well contains MTT in the range of 100-125micrograms.

After incubation for another 2-4 hours , you can very well centrifuge to sediment the formazan crystals and get the reading by dissolving it in suitable reagent.

Isopropanol (isopropyl alkohol) is a good solvetn for MTT formazans bur addioton of HCl makes it better. (60uL 6N HCl pro 10 ml Isopropanol). You can pipeate for better solibility. If you what to keep longer only in refrigerator in darkness.