There are limitations to most assays. and certainly several problems using the MTT and related assays. If you dig around,  you'll find some threads that go into this here at Research Gate, so I wont reinvent those discussions here. Viable cell counting using trypan blue dye exclusion and a hemocytometer is probably the least technologically demanding and the cheapest (but it can be a pain since its time consuming). Its reasonably accurate. Cell counting using a Coulter Counter is quick and reasonably accurate but doesnt always separate alive from dying cells. Crystal violet staining and reading optical density is cheap, quick and effective too as a means to measure changes in cell density. You can also measure 3D colony forming ability in methylcellulose or soft agar. If you search on this website, you will likely find other and more detailed responses to this tye of question. Good luck with the research...Bob

MTT is nowadays worldwide recognized as viability test also considering the limitations of the method. There are a lot of methods to assess viability. In my experience MTT or MTS are the easyest. I could suggest the sulphorhodamine assay (it is a dye binding to proteins) or methods based on DNA. MTT conversion is linked to mithocondrial function so it is possible that your treatment enhance mitochondrial activity without proliferating effect. You could assess mitocondrial activity. Moreover, a quick and convenient way to verifiy if there is a real proliferation effect is to observe the cells with a microscope and see if there are more or less cells in the treatment wells compared to the control. You could see this event at the first sight without counting the cells.

I’ve attached a couple of papers about CNT interference in the MTT assay, and given some info below about interference assays in in vitro cytotox experiments.

It’s quite common, and probably recommended, to try and use two functionally different assays for cell death. Common ones are measuring the mitochondrial activity of metabolically active cells (MTT, CTB etc), and measures of membrane impairment where you measure the release of enzymes such as LDH or AK that have leaked through damaged cell membranes into the supernatant. Also it’s common to use a positive control that will clearly induce cell death in your samples. Things like camptothecin can be used to induce apoptosis, but this would only really be used if you were specifically looking at apoptosis. Something such as Triton X-100 or saponin can be used to lyse the cells, so is a good control for both mitochondrial function and membrane impairment assays

 MTT interference

In the MTT assay the reagent starts as yellow (I think), there is the formation of an insoluble compound (formazan) inside metabolically active cells (viable cells). To read this change spectrophotometrically the soluble product needs to be made insoluble, which is done by 2-propanol. Test substances could infer with either of these steps by binding to MTT reagent or products, which would lead us to believe the materials are toxic when they’re not; or materials impact on the absorbance readings at the wavelengths that are used, which could hide any toxic effects.

Controls to check for this interference would be:

Adding the MTT reagent and test materials simultaneously to the cells, if the materials bind to the reagent, it would not be available to the cells and it would appear that there is reduced viability when there shouldn’t be.

Adding the Test substances (TS) after the cells have been given time to solubilise the MTT salt (maybe 30 minutes), if the TS bind the coloured product before it is made insoluble it would also appear there is reduced viability when there isn’t any.

Adding the TS alone (no MTT reagent) in cell culture medium and reading the absorbance at the wavelength for the MTT assay, if particles interfere here you could find that potentially toxic effects are hidden. 

You could also check if TS reduce MTT by themselves with no cells present by incubating particles with cell culture medium, no cells, and following the MTT protocol. But this may be a little excessive.

For other assays of mitochondrial activity e.g. CTB, MTS, alamar blue similar tests can be done. The ones mentioned here don’t use an intermediate step though, so the first two tests above would be done as one.

LDH Adsorption

There are a number of enzymes that can be assayed for that may appear in the exposure supernatant, common ones to look at are LDH or AK. Proteins can bind to test substances so it’s important to check if it’s occurring with your test substances

To do this you can incubate cells with 0.1% Triton-X100 for one hour, collect the supernatants and use this to suspend, disperse and incubate your particles (at experiment relative concentrations) for maybe four hours,  then spin out the particles and follow the LDH (or other) assay. If the particles have bound the enzyme there wouldn’t be any left to appear in the assay and it would appear that there was no cell death, when you know there has been as you have treated the cells with Triton. 

But i think you used microglia cells , i would suggest you to check with two differemt assay systems. Sometimes  metabolic activity will be induced by activated microglia in this case better to check LDH cell leakiness as well.

I hope this helps you somewhat? Let me know if you need anything else.

Hi, to my experience, MTT, Tryphan blue exclusion and LDH assays are cost effective and give expected results. And how do you get 150%? Have you counted the cells before you seed? 

I think you are inducing apoptosis in your treatment. Apoptosis induces mitocondrial activity.

Thank you for your answers. The answers were helpful. trypan blue, of course is the cheapest and most convenient way. I will perhaps pick it up along the line.

I was also wondering if i must go for assays like BRDU incorporation, or PCNA expression in my cultures to further validate the study. 

Just few Clarifications.

MTT is an established proliferation assay, so induction of apoptosis can be ruled out, and proliferation can be considered. Also, i have done a phase contrast study which demonstrated proliferation in my cultures.

The seeding density was same for both the cultures.

My question targeted the graphical representation more,than the functional significance of the assay. i wondered if representing it as 150% as compared to normal is a better way, or should i present the graph of the absorbance directly.

Many thanks again!

@Murali.

thanks. yes,  those things are taken into consideration, the same treatment induced cell death in neurons in our hands, so interference is not an issue. it is with microglia that we see proliferation, in reproducible experiments with this treatment. we are linking it to microglial activation mediated proliferation.

Yes Pooja as i mentioned MTT assay is a metabolic assay. In your case microglia activation may produce such increased metabolic activity.

But try to check the cell membrane rupture i mean LDH assay with the same supernatent. All the very bests

It appears that the treated cells are growing faster than the normal cells (if you see more cells relatively in the treated cells compared to control- under microscope.  In this scenario, you can blot either percentage or absorbance graph.  To confirm this you may want to do colony forming ability assay.  This is inexpensive.  Just treat the cells (6-well plate in triplicate) for set time points and then remove media and add regular media for about 2 weeks.  Stain the colony and take a picture and also count them compared to control.  You should see increase in either colony size or number compared to control. 

For the graph, I encourage plotting the vertical axis based on what is actually measured, namely absorbance. However, sometimes the data are difficult to visually compare and setting the time zero to 100% and plotting the percent signal is acceptable.

If you use percent, be clear what the percent refers to. It is _NOT_ percent proliferation or percent growth (or for a decreasing signal, percent death). Instead, you are plotting percent of initial absorbance. Any other axis title is based on interpretation of the data- this should be saved for the results/discussion and not for the figure axis.

The MTT signal will always be indicative of multiple processes, including proliferation and death. The axis label should not serve to obscure this balance.

Thank you for your valuable answers. i will try taking into consideration the colony forming assay and LDH assay.

@ Dr. Justin Mott, I fully concur.  OD is the apt representation, and i will go for that. In the other case, we reperesnt the value as %viability, so if Noral control is 100 %, the cells in treated scenario are 1.5 folds more (150%). For now, i ll stick with OD values only. Thanks much, your answer was very helpful.

No. % viability means to discriminate live and dead cell usually based on permeability of cell membrane. This measurement fails to uncover early apoptotic cells which are in fact dead (but have intact membrane). This is really huge problem! On the other hand, MTT assay is not able to distinguish between the number of cells and the decreased viability. The decreased value of MTT reflects either the decreased number of cells or the cell death, or very often both events. However without further analysis you do not know what has happend.  

Petr