Hi,

It will depend on the kind of cells you are working with. If your cells detach easily from the plates (like mine) it will be better to leave the MTT in the samples. Just dissolve the crystals with DMSO and then read the absorbance at two wavelengths: at 562 (or 570 nm, depending on the filter in your microplate reader) and at 620 nm for reference. Then you'll have to subtract the absorbance at 620 nm from the absorbance at 570 nm, and use this corrected value for your calculations. In this way you won’t loose cells.

Best luck!

Sandra

We usually remove MTT before adding to DMSO. We do that to remove the unbound MTT that might interfere with the spectophotometric analysis.

@Noor Nordin: I agree with Kiran. It is advisable to remove all the remaining unbound MTT prior to DMOS addition to avoid any nonspecific absorption and high signal to noise ratio. Bests

I know I'm not answering the question, but as a suggestion, switch to MTS instead of MTT. Best of luck!

Removal of MTT should not be done because whatever formazan crystals have been formed are a result of cellular mitochondrial dehydrogenase enzyme reaction. I believe that removing of MTT will resulting in loosing a significant amount of those important crystals and this may vary your analysis because the cells die during the MTT conversion to fomazan and if non-adherent then a number of cells having crystals will float in media and aspirating that media will also take those cells out. So, let it be because for non-specific absorption one can normalize the absorbance range from 450-570nm (MTT) with reference wavelength 630nm.

I would never suggest of aspirating anything after MTT incubation is over. Use separate only media wells and media + vehicle (e.g. DMSO) in triplicate to normalize the absorption values by subtraction.

One can think about the suspension culture cells where MTT crystals would be floating in the media now what could be the possible way out? Do those suspension culture would not show so called non-specific absorbance? It means we must centrifuge the whole plate and then continue for DMSO? Will such centrifugation is not going to precipitate everything or still the left out crystals or dissolved crystals which will be aspirated are not significant for the study?

I hope Noor you have to think about it. Good luck

Yes, you have to remove the MTT medium before adding DMSO. To be sure you have to revise all the official protocol of MTT assay.

It is better using the related compund WST-1, or any of its upgrades. This has better signal-to-noise ratio, and you do not have to solubilize the formazan crystals by adding DMSO! An even better (and cheaper) compound to use, is resazurin (Sigma) which requires a fluorometer, but has excellent signal-to-noise ratio and is almost non-toxic for the researcher as well as for the cells :-)

I agree with the other responses, you need to centrifuge your sample to collect the crystals and remove the medium. This ensures that you are dissolving the MTT crystals in a know volume of liquid. Further, I am not even sure that the MTT crystals will dissolve properly if you don't remove the medium. Also as stated by others there are many alternative stains to MTT that don't require removal of the medium: XTT, WST-1 and Alamar Blue. The disadvantage of these is that they are more expensive, if you do decide to try one of these other stains it is advisable to use phenol read free medium. Good luck.

Hi,

It will depend on the kind of cells you are working with. If your cells detach easily from the plates (like mine) it will be better to leave the MTT in the samples. Just dissolve the crystals with DMSO and then read the absorbance at two wavelengths: at 562 (or 570 nm, depending on the filter in your microplate reader) and at 620 nm for reference. Then you'll have to subtract the absorbance at 620 nm from the absorbance at 570 nm, and use this corrected value for your calculations. In this way you won’t loose cells.

Best luck!

Sandra

It is necessary to remove MTT medium because the reaction product formazan of MTT is not soluble in aqueous media and this is why organic solvent such as DMSO is added after. However, the formazan product of MTS reduction (MTT derivative from Promega) is soluble in cell culture medium and does not require use of an organic solvent. Samples are read directly after a 4-hour incubation.

Hi,

We always remove the medium before adding the DMSO. We do this for both suspension and adherent cell lines. We just make sure to spin the cells down before media removal. Once media is removed formazan crystals can be dissolved in 100 uL DMSO and plates are then put on a shaker for a few minutes to ensure that the dissolved formazan spreads evenly through the well. Media creates alot of noise and dilutes the purple formazan colour so i wouldn't advise leaving it in the well.

Good luck =)

Hi Noor,

It depends on the cell type (adherent vs suspension) & on your drug as well because some drugs might interfere with MTT-formazan formation or promote the conversion chemically resulting in an overestimated viability. My suggestion is to try both ways & compare the results. Correcting the readings as proposed by Sandra or trying other soluble version of MTT, such as CCK-SK from Dojindo which worked well with me are also possible options. Alternatively, you can use a "triplex" solvent system to dissolve the formazan crystals without the need to remove the media (99 mL DMS + 1 mL 1M HCl + 5 g SDS).

All the best.

Hi.

I think that remove the medium (gently) is better. because medium interfere with last color.

In general, MTT crystals are not soluble in cell media. When working with adherent cell lines, after MTT crystals are formed, cell media has to be aspirated gently prior to adding DMSO. For non-adherent cells, spining the plate down is an option or collecting your cell samples in tubes and spining them down.

If you add DMSO directly to cells in media, it is advisible to use a larger DMSO volume (double the volume) to make sure all the crystals are dissolved. This can result in diluted formazan colour, which is not always good for your measurements.

If possible, use of stains that are soluble in cell media is advised.

I agree with most of the suggestions. It is important to remove the medium before adding DMSO. For strongly adherent cultures this can be done directly. For suspension cultures one needs cetrifugation. The loss of cells that supposedly die and float, as mentioned in the first comment, should not be significant, if at all. The cost of losing the signal due to insolubility of formazan and its dilution weigh more than losing a few cells. Morever, it is expected to always run standard curves with graded cell densities and this should limit thd errors too.

You could consider XTT tests where you just add Xtt reagent. The product created can be read out directly. You don't need to dissolve it.

I agree with DAVID and SANDRA. u do need to keep MTT in culture medium.

I agree with DAVID, SANDRA and Lalini, you can to keep MTT in culture medium .

Removing the MTT medium (after plate centrifugation) does reduce background noise but it must be done carefully if you are not to lose MTT signal also (why some do it on an individual well-by-well basis rather than multi-pipette). For that reason I have gone with the Promega MTS assay (as advised by Hamid) and used double the volumes suggested in the protocol. The kit comes with a STOP/Solubilisation reagent which you add after the 4 hr MTS incubation. I leave it overnight before plate shaking and absorbance reading but there is a quicker method.

Thanks for all the inputs, I really appreciate that. I am really glad to learn those reasons behind each one of the arguments, eventually these will guide me in preparing my lab works.

I follow a protocol with adherent cells in a 96 well-plate, and it says that after incubation with MTT you have to remove all medium but 25 ul, and then add 50 ul of DMSO. Then mix and incubate 10 min at 37ºC. Mix againg and change the supernatant to a new plate because I think that maybe the cells on the surface can interfere with the measure. I use the 540 nm filter in a microplate reader.

We used to remove the medium (carefully) and replace with an aqueous solution of 10% SDS and 50 % DMF. In recent years, we have migrated to Dojindo's CCK-8 assay, which is based on tetrazolium salts similar to XTT and WST-1. No removal step is needed, and absorbance can be measured repeatedly without sacrificing the culture (i.e., if signal is not yet in the good detection range for your plate reader, continue the incubation and read again later).

WST reagent is very good and does not require DMSO step. You can do 800 test with 8 ml solution. Its available from Roche diagnostics.

MTS, or any media-soluble formazan varient, is much easy to use that MTT, although more costly. I've used the "all-in-one" formulation (CellTiter96AQueous PMS + MTS), where you just add the MTS solution to phenol-red free media (that is very important). No stop solution is required, but it is optional. You can either add an aliquot to the existing media (if you're worried about aspirating cells), or change the media and replace with phenol-red free. Regardless, you could incubate as long as necessary until sufficient color develops- unlike the "blind" MTT method, where as soon as you add DMSO, you're stuck with whatever the cells have converted. Also, it's crucial to remember that all you are measuring is a cell's ability to reduce a chemical, which means they can't have consumed most of the sugar in their media, which means that cell plating density and treatment duration/media changing schedule are all things that have to be controlled. Perhaps optimizing the assay conditions would be more efficient, faster and more reliable with MTS, and then if you have to run numerous trials at similar conditions, you could switch to the cheaper MTT?

Cell medium needs to be aspirated as the formazan crytals are not soluble in cell medium, but are in DMSO. When aspirating, care needs to be taken not to remove formazan crystals. Alternatively, DMSO can be added in access of cell medium, however it needs to be determined experimentally. I'd suggest you start with 1:2 ratio (twice the volume of DMSO).

Answer from Miriam Funes seems a nice option, leaving a small volume of cell medium and adding twice as much DMSO.

For MTT, you should absolutely remove culture medium before adding DMSO. You do not need to remove culture medium only if you use MTS from Promega.

Methyl tetrazolium (MTT) assay

The protocol was based on a method described by Plumb et al. (1989) using MTT dye. This assay is depends on the cellular reduction of MTT dye by the mitochondrial dehydrogenase of viable cells to blue formazan product which measured spectrophotometrically. Briefly, after culturing CHO-K1 cells in a 96-well tissue culture microtiter plates at a 65% confluence, the medium was replaced with serum free medium containing the different concentrations of the tested compounds. After 24, 48 and 72 h of exposure, the medium was removed and immediately 50 ml of MTT dye in PBS at a concentration 5 mg/ml was added to each well. The plates were wrapped in foil and incubated for 4 hours at 37oC, the solution of MTT in PBS was removed and 200 ml DMSO per well was rapidly added, followed by 25 ml of Sorensen`s glycine buffer (0.1 M glycine plus 0.1 M NaCl equilibrated to pH 10.5 with 0.1 M NaOH). After rapid agitation of the plates for 15 min at room temperature, the optical density of the blue formazan product was measured using an ELISA microplate reader (Diagger model JX8613) at 570 nm.

In my experiance removing the medium (by decanting the plate ) increses the solubility of crystals.

In my opinion, we used to remove the medium containing the MTT before adding DMSO, because MTT itself has color, which might interferes the absorbance readings. Also, the crystals dissolve better without the medium.

In place of MTT you can perform SRB assay as SRB relies on relies on the uptake of the negatively charged pink aminoxanthine dye, sulphorhodamine B (SRB) by basic amino acids in the cells whereas MTT on are metabolically reduced by the mitochondria in viable cells to a coloured formazan so, if the function of these is inhibited by variations in cellular levels of NADH, glucose and other factors, variable results are obtained and a similar result may be given as if the cells were not alive or not proliferating and if it is only the case of solubility then The use of XTT is preferred since the formazan produced is soluble in water and the solubilisation step required if MTT is used is eliminated

Dear friends, please I need your help

I have problem. I treated my cell with quercetin and green tea separately. I used MTS, Neutral red uptake assay and crystal violet but no results. The problem I get DO values similar for the different doses and for the highest dose I got OD such as the control so I can not calculate cells viability to get IC50. I conclude the problem is due to the precipitation of these compound on the cells which will interfere with colorimetric reading but I can not solve this problem. I found many articles mentioned using theses assay fro these compounds but how the get results? Some of them mentioned washing with PBS to wash but I tried it and doesn't work. These compounds has reducing properties for MTS but suppose to work with Neutral red uptake assay

Your help is highly appreciated

Thank you so much for all the valuable comments, they were really helpful in making me clear with my methods. As for Mohammed Nashiry, maybe you should try freeze-drying your aqueous tea sample and later prepare them fresh in your favoured concentration, by dissolving the freeze-dried tea powder just using the culture media (if you haven't try this yet). As for quercetin, I've never used them before so have no idea and experience.

You might be better remove the sovelable MTT in order to avoid the false positive OD.

In my experience, removing the medium containing the MTT before adding DMSO is better, because of two reasons mentioned in above comments; formazan crystals are soluble in DMSO and not in media. Secondly, yellow colour of MTT can interfere with reading.

I usually remove the media containing MTT first before adding DMSO. Agreed with others as mentioned above.

In my experience, aspirate the medium containing the MTT before adding DMSO to avoid interference during Do reading

In my experience, aspirate the medium containing the MTT before adding DMSO

Im doing MTT for past 4 years almost everyday...

1. I dont remove MTT from the media, If im using DMSO, I ll take the reading immediately after 10 mins.

But almost all the time I use 10% SDS acidified with 0.1N HCL to dissolve the formazan crystals,after 4 hrs of MTT i add SDS and keep it overnight, next day morning i ll take OD and do the viability. SDS is more efficient than DMSO in context to stability of the purple color.

Hope u r adding MTT to blank wells containing media alone to avoid the background!

Why not try MTS assay which is much more convenient, and there is no need to change the media and add DMSO

YES removing the MTT medium prior to DMSO is essential for accurate results.

Yes.. I did mistake once and I learned my lesson..

We do a lot of cytotoxicity and proliferative assays and with MTT after 2-4 hours of incubation with MTT we just add 100 ul of acidified isopropanol to the wells (isopropanol containig 0,04 N HCl) and mix with a automatic pipette a few seconds or on a shaker for a few minutes. Acidified isopropanol dissolves everything very nicely and you got a very uniform clear colour, so the blank is nicely yellow (only MTT with isopropanol, no cristals) and the proper samples are brownish-purple (purple formazan with yellow MTT and transparent isopropanol). We usually read the test at 630 nm or 570 nm depending on the plate reader and we have no problems. Nice, easy and cheap.

Removing media before adding DMSO is necessary to avoid back ground. However, media should be removed gently to avoid removal of formazan crystals while pipetting out.

I agree with the rest of people. I think the removal of media with MTT is necessary before adding DMSO to dissolve the precipitated crystal.

removing the media is required before adding DMSO to avoid any interference with the leftover media... in the final results

I would reccomend the removal of media beore adding DMSO. it gives good result

yes, from ,my experience removing media is very important to avoid interfering to measure OD as well adding DMSO IS NECESSARY for dissolve formazan crystals . and i recommended use the shaker for 10 to 15 mins and read at 520 nm

I used to remove the medium but later I tried to leave it and the results was the same, so if you want you can leave it.

Yes remove

In addition to adding DMSO, you also can add other solutions directly to dissolve the precipitated crystal without medium removal .

Hi I agree removing mediem is ideal- but extreme care should be taken to not interfere with formazonand cells.

Variations in the OD generally comes with removal of media so in that case a different solubliser can be added like 10% triton in 0.1 n HCL in isopropanol. It dissolves crystals in a better way. I tried it with suspension cultures where removing media is not possible.

Hi I have been reconsidering the issue of removing media. If we realise that we are working with nanoquantities of enzymes and their effects even the slightest interfnce with media may affect the accuracy. Thus l would not remove media.

Might be a bit off topic, but if removing cell media is not an option, other agents, such as Presto Blue can be used. Presto has an advantage of producing soluble crystals in cell media, thus removal of cell media is not neccessary, and both fluorescence and absorbance can be measured, also incubation time is shorter.

i believe that by removing MTT medium you can get better result.in addition for controling PH you can use sorrento buffer.

Its advisable to remove the medium to ensure the complete and uniform solubility of formazan crystals. But working with certain agents like plant samples or nanoparticles, where the disturbance of culture medium disturbs the crystals at the bottom, the reproducibility is higher without replacing media.

What concentration of DMSO do u use to disolve the MTT crystals? and have u ever heard about the use of formalin to dissolve MTT crystals?

Removing mtt media before adding dmso gives good results.l am doing this in cytotoxic assays of tobacco extract on lymphocytes.

I agree with the above answers. What if cells get detach under treatment conditions of drug/ NPs? In that case removing media will be suggestible? 

When working with non attached cells like [ymphocytes , not removing MTT before adding DMSO gives reproducible results. Could somebody explain the rationale of taking OD at 620 AND 570nm

Hi

Removal of media before addition of DMSO results in proper dissolution of formazan crystals, which is affected if the aqueous media is left there (formazan crystals are organic solvent soluble).

For non adherent cells you can centrifuge to pellet down the cells. this will result you in a reproducible results.

Regarding 570/620: %70 is the wavelength used to record the absorbance due to the purple color of soluble formazzn and 620 is used as the reference for the yellow color of media interference (when you remove the media before addition of DMSO at that point only 570nm reading will be fine need not to bother about the 620). The wavelength of 620 is important when you leave the aqueous media or when we use isopropanol/HCl method to dissolve the formazan crystals.

Thanks and good luck happy working

Anil

Hi

The removal of media with MTT should be complete?? for example, if I have 200ul per well, can I just remove 150ul? Because, I am afraid of loosing the crystals...

Thank you

Leticia

Hi, in my experience could work but cost a little bit longer to dilute. At the end i resolved the problem using instead of dmso a solution composed by

isopropanol, 10% Triton X-100 and 0.04 N HCl. worked very good but always with a blanck

Thank you!!

I have another two questions:

1) Is it possible the absorbance of testing samples is higher than control? Given a viable cells like 120%?

2) Someone read the absorbance in 490nm? Because my plate reader just have this option, and I saw in some papers this wavelength.

Thank you again

Leticia

1) Generally, the control it's an average of several replicates. If your tested samples are having an absorbance higher than the controls, the tested samples might be promoting cell growth. You need to certificate if such characteristic of your sample it's important to regard in this step of your experiments. If not, you can evaluate the cell growth of this substance in future experiments.

2) I always do my reads of absorbance at 540 nm. But I already read several papers that did in 490 nm. So, I think that is not a problem for your assays.

Yes it is necessary to remove the MTT medium

In relation to:

2) I always do my reads of absorbance at 540 nm. But I already read several papers that did in 490 nm. So, I think that is not a problem for your assays.

- Probably is MTS not MTT.

Depends upon the nature of cells you are using. To be on safer side its better to remove the MTT containing media to avoid any unbound fractions of MTT in the media. 2nd crystals are not soluble in media and it can also interfere with the absorbance so its better to remove and then add DMSO.