Dear Swayam Ma'am,

For best use, dissolve 5mg MTT powder in 1ml PBS (cover with aluminium foil). from there, just take 20ul for each well of 96 well plate format. It works nicely, avoid chloroform.

Thanks & Regards

Saurabh

Dear Saurabh,

I am aware of the MTT assay protocol. MTT powder is yellow in colour and dissolves quite well in PBS. MTT- formazan end product is purple in colour and dissolves only in chloroform.

This is the whole problem.

Mam, to dissolve the formazan crystal, did you tried DMSO?

Yes it's soluble in DMSO. But what's the point? The amount of formazan formed relates to the cell viability. When u are already providing formazan, how it will be related to viability study?

Mam, MTT is a colorimetric assays, bit tricky for analyzing cell proliferation/viability.

We all know, it is generally used assay but less sensitive compared to other available assays.

It is cell number which is determining factor. The healthy and rapidly growing cells exhibit high rates of MTT reduction to formazan while the dead or inactive cells fail to do so. The final product of MTT reduction is a purple color formazan that can be easily dissolved in DMSO.

Viability in the MTT assay is connected with the quantification of formazan at 540 nm which is linearly associated with the enzyme activity and indirectly the number of viable cells.

High purple color intensity denotes higher cell viability while the decrease in purple color intensity signifies the reduced cell number and thus cytotoxicity of the given substance.

Hello,Step 1. you make a series of dilutions (for example 5xE-1, 2.5E-1, 1xE-1, 5xE-2, 1E-2, 1xE-2 cells/mL) with known cell density in triplicate at least, then add MTT in PBS buffer, incubate for a certain time, remove unreacted MTT and then dissolve formed formazan by propanol 0.04M HCl(or DMSO),

Step 2. you plot the data absorbance vs cell density calibration curve.

Step 3. Using a calibration curve you can obtain a data for cell density with unknown concentration.

Finally, chloroform for extraction of formazan is a very bad idea, as it evaporates fast and data is not reliable. More information in attached paper. This method equally well applies for mammalian cells and bacteria.Article A cryogel-based bioreactor for water treatment applications

I have the same question, I know mtt-formazan its de finally product, so i cant use to evaluate viability but... what use has mtt-formazan? I think i loss money

No, you did not loss money. MTT has tetrazole structure and due to cell's metabolic activity it gets converted to formazan this compound does not have tetrazol cycle and has violet colour and it is soluble in DMSO and propanol. please follow the instruction above

I know all about MTT...

But I dont have MTT, im talking about MTT-formazan (MTT- formazan (Sigma) is a purple powder, soluble in Chloroform (10mg/ mL)).

Sigma sell this product but is the final product... so I dont know what is the use.

And the product dont have Instructions or description about the use...

I also received formazan and struggled. I never thought that it would be a purple powder. It is 5 bottles. I think I lost the money.