Usually, you would prepare a 5 mg/mL solution of MTT, which would be your 10X stock solution. What brand is your MTT?

I am using it from HiMedia

Read this...it contains full information......

OK, so you should prepare a 5 mg/mL stock solution. So, in a 96-well plate, it should go like this:

- add 90 uL of culture medium to each well (Be sure to use a medium without phenol red, as it interferes with the reading. You could use PBS as well)

- add 10 uL of your MTT stock solution and wrap the plate in aluminium foil to prevent exposure to light.

- after 2-4 hours of incubation (you need to set this up as it is highly variable with exp conditions and amount of cells), check until the precipitate is clearly visible.

- Now you need to solubilize the dye, so you have to remove the medium and add 100 uL of solubilization solution -Acidified isopropanol (0.04 M HCl in absolute isopropanol works fine, but there are various adapted methods)-, incubate for 15-30 minutes with gentle shaking (make sure the crystals dissolve completely).

- Read at 570 nm.

This is a basic protocol that applies if you're using adherent cells; if not, you need pellet the cells.

Hope it helps,

Daniel got the complete answer. I incubate for 3 hrs after adding MTT in 37 degree centrigrade and as alternative to Isopropanol you can also use DMSO for dissolving precipitate.

Thanks Daniel for your kind reply.

Also thanks to Maaz for the article.

Usually I do MTT assay after the wells get fully confluent. I use the same stock dilution, i.e. 5 mg/ml. But media I use is DMEM with 1% FBS in case of experiments.

I add 200ul of MTT in each well, incubate it for 3.5 hrs in dark and then discard MTT, dissolve the precipitate in DMSO and record the absorbance at 550nm.

I use HepG2 cell line.

Does it need any modification?

FBS should be 10 % in media.

1. MTT SOLUTION is stable when stored frozen. Storage at 2-8 °C may result in decomposition and yield erroneous results. Development of dark color or formation of crystals indicate product deterioration.

2. Microbial contamination will contribute to the cleavage of MTT and formation of MTT formazan yielding erroneous results.

3. Uneven evaporation of culture fluid in wells of multiwell plates may cause erroneous results.

4. High protein levels ( serum, albumin, etc.) in the culture medium may form a precipitate when MTT SOLVENT is added. Samples with protein concentrations equivalent to 10% fetal bovine serum seem acceptable. Sera with higher protein

concentrations than fetal bovine serum may have to be used.

During passage, we use 10% FBS, but during experiments, we use 1%. Is it wrong?

Adaptation!

5mg/mL stock solution of MTT should be store at dark and cool place

then you can dilute your stock to 0.5 mg/mL

10x MTT stock solution can be prepared at 5mg/mL and stored at -20C for up to 3 months. 1x MTT working solution at .5mg/mL can be stored at 4C for up to 1 week. Avoid light and rapid thaw. DMSO is a great solvent; also, put cells on a shaker at high speed to assist in DMSO solubilization of formazan crystals.

Hi Danial,

Just to confirm. DId you add 10 µL of the stock MTT (5 mg/ mL) to the well or the working concentration ( 0.5 mg/ mL) to the well.

PLease anyone could confirm this would be appreciated.

Thanks.

You will need to add the working concentration (0.5 mg/mL) for the assay. Stock solution (5mg/mL) will most likely kill the cells.

Best of luck.

Thank you Guys for this conversation ;) I was looking for the working concentration of MTT reagent... and now I know:)

All the best 

hello - is the MTT formazan product in the cells or outside - if inside do i sufficiently lyse the cells to have all the dye in solution ? lg m

Dear Martin,

the DMSO will do the work. Once you add the DMSO, the formazan will be dissolved and you probably need to put the plate on a shaker for 5-10 minutes to assist the dissolution of the formazan.

Hope this help.

Thanks.

hello, this helps very well thanks (: greetings Martin , if i have 375 mykroliter in the well and 20 mykroliter of mtt you think 50 mykroliter dmso will do the work ? the wavelength for Absorption should then be 540 or 570 nm, ?

Dear Martin,

I used a 96 wells plate and seeded 100 μL of cells/well in regular complete DMEM, allowed the cells to settle overnight. The next day, the cells were treated with the compounds that I am studying. After 24 h incubation, the medium was aspirated, washed with 100 μL PBS and later added with 100 μL of DMEM -MTT reagent ( 1mL of MTT stock (5mg/mL) +9 mL of phenol free DMEM), incubate 4 h and read at 570 nm. 

Hope this help you.

You can modifiy the protocol according to your experiment.

hi - i add dmso to the sample in each well after incubation at different time Points - 75 mükroliter dmso seem to work but mixig a lot with the Pipette and viewing optically first if everyting is dissolved is important , i will see how it works with greater bacteria concentration at later time Points as i have just place for about 100 mykroliter dmso in one well greets Martin  

Yes, 

Danial and Yong are correct. I forgot to include, after incubation (3-4h), aspirate the DMEM, make sure to be gentle so that the formazan is not aspirated together with the medium. If you use phenol red DMEM, better add washing step first before adding the solvent. If not, add 100 uL of 1:1 DMSO: ethanol, put on a shaker for 5-10 minutes then read at 570 nm. 

the details is included in this pamphlet

The cell culture suspension is washed with 1x PBS and then added 30 μl of MTT

solution to the culture (MTT -5mg/ml dissolved in PBS). It is then incubated at 370C for

3 hours. MTT is removed by washing with 1x PBS and 200μlof DMSO is added to the

culture. Incubation is done at room temperature for 30 minutes until the cell get lysed and colour is obtained. The solution is transferred to centrifuge tubes and centrifuged at top speed for 2minutes to precipitate cell debris. Optical density is read at 540 nm using

DMSO as blank in an ELISA reader (ELISASCAN, Erba). The cells are also observed

using fluorescent microscope (CKX41).

The cell culture suspension is washed with 1x PBS and then added 30 μl of MTT

solution to the culture (MTT -5mg/ml dissolved in PBS). It is then incubated at 370C for

3 hours. MTT is removed by washing with 1x PBS and 200μlof DMSO is added to the

culture. Incubation is done at room temperature for 30 minutes until the cell get lysed and colour is obtained. The solution is transferred to centrifuge tubes and centrifuged at top speed for 2minutes to precipitate cell debris. Optical density is read at 540 nm using

DMSO as blank in an ELISA reader

Kunal Pal, this time you copied and pasted Hasna Abdul Salam's answer.

Hello, I have a problem with MTT assay.

After seeding the cells in the MTT plate after 72 hours, they are all dead (even in the control). The cells are round without their characteristic shape settling to the bottom of the wells.

Could it be that something is wrong with the plates? Perhaps you can share how you prepare cells for the MTT test?

Silvija Venyte can you give more informations about your protocol? What cell line are you using?

E lecture for MTT assay https://youtu.be/gnj5ULsSNOo