Hi,

You have to keep the following things in mind:

a) Instead of using a kit, just buy the MTT powder and use that for your assay. Buy from Sigma.

b) If you don't see many crystals after hours of incubation, something is wrong about the MTT or you don't have enough cells in the well.

c) After incubation of cells with MTT, remove all the medium and add DMSO. Only then you will observe purple colour.

Good Luck!

If all reagents were prepared in accordance to manual I could be problem of cell viability or quantity (12000 could give very weak signal).

In our experience we using DMSO:ethanol  (1:1) instead of Crystal Dissolving SDS. It working very good (fast and effective). You even don't need to transfer any solution - just measure OD570 and subtrack OD700.

To be sure about formozan formation it very easy to observe cell under inverted microscope. Small dark dots and sometimes violet crystals will appear in vial cells after 1 hr, if cell metabolically active.

Incubate MTT with your cells for 4 h at 370C is more than enough. Utkarsh is correct that you have to remove all your media and add DMSO to see purple.  Otherwise, you'll only see light yellow (the color of MTT).

Thank you for all your suggestions! 

I'm working with PBMC cells (lymphocytes) so would DMSO still be useful to dissolve the crystals? 

The kit said to seed anywhere between 500-500,000 cells so I seeded 12,000 which I thought would be plenty. The cells are also almost all healthy because I made sure to count them underneath a microscope in which I saw almost no dead cells. 

I phoned Cayman chemical's technical support so hopefully I will have a solution soon! Thank you! 

Hi Suzy,

To do mtt assay ,I usually  add acidified isoprpanol i.e 250 ul concentrated hydrochloric acid  in 100 ml isoprpanol to dissolve the formazan crystals which are clearly visible under the microscope after 4 hour incubation with MTT

Dear Suzy,

I've never used kits for MTT assay because every researcher should be able to dissolve tetrazolium bromide (MTT reagent) powder in a few drops of alcohol and distilled water with following 0.2 mkm syringe filtration. And DMSO is available in any cultural laboratory shelves. Reading what you did I can say that if you want reliable results of survival/toxicity 1) MTT is for 48-96 hours of incubation applied after 12-24 hours since you passaged your culture into 96-wells plate. 2) orbital shaker is not necessary, you can successfully mix MTT reagent gently moving the plate with your hand on laminar table for 20-30 seconds - cells survive better. 3) Several months ago I wrote detailed protocols for MTT assay performance on this Q-A area here:

https://www.researchgate.net/post/Can_you_answer_a_few_questions_concerning_the_Cell_Counting_Kit-8_and_the_MTT_method

and here:

https://www.researchgate.net/post/What_is_the_optimum_conc_of_a_test_drug_in_MTT_assay

4) It is not only cells survival but also their proliferation which lead to formazan crystal's formation in your assay. About primary PBMC - if you did not use their stimulation with cytokines IL2, PHA, etc. the active proliferation is not a feature you can expect from PBMC. For PBMC proliferation stimulation and differentiation blockage different cytokines sets are being used. If you are interested how to work with PBMC beginning from the isolation and ending with in vivo applications you can find detailed protocols in my patent "In vivo model for HIV vaccine efficiency evaluation..."  HIV vaccine was developed in my laboratory completely on PBMC primary cultures background.

Hope both info might be useful for you. MTT is a very good method which expert-level handling can give you the abilities from a testing almost every water-soluble agent biologic activity to a measurement of alterations in a single protein phosphorilation in vitro.

Best regards,

E.F.

I agree with Utkarsh Kapoor and I also recommend the MTT powder from Sigma! It has always worked for me. With regard to using DMSO after removing the medium, it is totally the right way to execute the method. 

Please, tell us if it worked. OK?

Best regards,

L.F.B.B.

Why don't you try more cells? I think 12,000 cells are not enough to get a positive signal. I usually seed at least 50,000 cells per well in a 96-well plate. I once seeded less cells, and got the same result as yours.

Dear students,

as I wrote above it might happen that with PBMC - depending on passage after isolation and their treatment or its absence - IT might not work at all. And nothing strange if so... Cells number per well does not matter.

Thank you all so much for your responses! 

Unfortunately, there was actually something wrong with the kit but I will definitely take your advice to hand for my next MTT Assay! 

I fully agree with Utkarsh Kapoor's answer. 

I think your mtt solution is not working. you can try with other cell lines to check whether the kit is working or not. 

 Hello,

It seems that there is something wrong with your MTT.

But in general there are several cell lines and especially AML cell lines that require a higher cell density/ well (for sure higher than 12000/well) to form crystals with MTT and then to give you the purple color.

Hello Suzy An,

How about your MTT assay? I have the same problem, not change purple color. Could you tell me, where is wrong, Please!

As was stated before:

1. Test your kit with any available cell (adherent is preferred) - You should able to see purple or deep blue points in cell cytoplasm after 20-30 min of incubation. Theoretically for some cells it could take up to 4 hr but in our experience 20 min is minimum.

2. If you working with suspension culture be sure what you not lost your cells before formazan extraction. F.e. spin down cells prior to removing media and addition of solubilizung solution. If methabolic activity is not very high then cell layer will not be purple, but then you add DMSO or DMSO:EtOH (1:1) you will see color.

3. Check your culture media and other cell contacting media pH - may be you kill cells before conducting MTT-test. MTT working solution must be yellowish.

Have a nice day and experiments!

Danill

I had the same expericnce today following using the abcam MTT protocol. The colors were pale yellow, not purple, however the results (ratios of PHA and CD3CD28 activated to the untreated wells) were rational! I think Utkatsh and Ying are right, and It is because we do not remove the supernatant following 3 hrs incubation by centrifugation and consequently the formazan colour can not be detected by the eyes.

http://www.abcam.com/kits/mtt-assay-protocol

Hi,

I am experiencing something vice-versa, means I am getting uniform purple color in all the wells. Which means I am expecting yellow color in wells which has complete CPE. I could visually observe in microscope that complete CPE is there but after adding MTT and incubated o/n in solubilization (10% SDS in 0.01 N HCl protocol) we could see only uniform purple color instead of yellow color in inactive cells.

Can anyone help me to resolve this issue.

Thank you

The "gold standard" approach is to have correct negative and positive controls (cell free wells f.e. with all testing concentrations of the substance). Some compounds (I don't know what CPE is) could give you color byself or affect the resulting color of the final reaction mix. Until this does not interfere with measurements on the wavelength of interest it is OK. But if absorbing spectras overlap it is obligatory to make corresponding corrections.

Even doing this it should be taken into account possible artifacts due to compound metabolization, oxidation and absorbance spectra change depending of pH.

In other words for the first experiment and assay development, it is better to check spectral properties of the compound of interest under conditions you going to use during your endpoint measurements.

I hope it will help to identify the issue.