MTT assay

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RockyDoc
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MTT assay

Hi,

I am planning on using a MTT-based assay for assessing cell proliferation/viability. Has anyone used the alternatives XTT or WST-1? If so, which of the three dyes is best?

Cynthia
Cynthia's picture
Yes, I have used all three.

Yes, I have used all three. WST-1 is the most sensitive of all three. It is supplied in a liquid form. If your choices are between the MTT and XTT--go with the XTT. You can save some steps with the XTT because it has been formatted so that the color change occurs while proliferation occurs. You will not need to add DMSO for the color change as you would need to do for the MTT.

vasipalli
vasipalli's picture
Hi. I have done MTT, if u

Hi. I have done MTT, if u want i could help u with MTT. just PM me

RockyDoc
RockyDoc's picture
vasipalli wrote:Hai

vasipalli wrote:

Hai
I have done MTT, if u want i could help u vth MTT.just PM me. Thanks but I have already completed the MTT assays. Instead of using the WST1 I used the MTT assay.

Many thanks for the offer of help.

naziasulthana
naziasulthana's picture
I want to do MTT assay for M

I want to do MTT assay for M NFS 60 cell lines for GCSF. I tried doing with 70,000 cells /ml after 48 hours treated with MTT and after 24 hours treated with acidified SDS but i didnt get gradation in OD ie all the OD are equal for 3 subsequent 10 fold dilution. I dont know why
Pls help me in solving this problem
Thanks and regards
Nazia Sulthana
vasipalli wrote:

Hai
I have done MTT, if u want i could help u vth MTT. just PM me
naziasulthana
naziasulthana's picture
want to do MTT assay for M

want to do MTT assay for M NFS 60 cell lines for GCSF. I tried doing with 70,000 cells /ml after 48 hours treated with MTT and after 24 hours treated with acidified SDS but i didnt get gradation in OD ie all the OD are equal for 3 subsequent 10 fold dilution. I dont know why
Pls help me in solving this problem
Thanks and regards
Nazia Sulthana
RockyDoc wrote:

vasipalli wrote:
Hai
I have done MTT, if u want i could help u vth MTT.just PM me. Thanks but I have already completed the MTT assays. Instead of using the WST1 I used the MTT assay.

Many thanks for the offer of help.

samm
samm's picture
Reduce cell numbers - ideally

Reduce cell numbers - ideally you should titre it. 70 k of those in a 96 well plate is very crowded.
Also, are you actually doing the MTT assay for 24 h?? That will virtually guarantee saturation - 3-6 h is a standard time (treatment with GCSF can be as long as you want, but to get the most info out of your expt, you should have a kinetic assay - 12, 24, 36,48 h of GCSF, followed by 3-6 h of MTT, followed by acid alcohol/acid SDS) - you would ideally need to do this first before titering the GCSF.

abhilash
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i need help with mtt assay as

i need help with mtt assay as wanna know about the procedure and the fullform of it..i also need somthing on apoptosis kita

Sum
Sum's picture
what cells are you working

what cells are you working with and which apoptosis kit ? Do you want to quantify apoptosis or is it enough to just detect if there is apoptosis occurring or not ?

Jason King
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Just a suggestion to tag on

Just a suggestion to tag on the end:

I'm using the Alamar Blue proliferation system and find that it offers a number of advantages. The main one is that you can take readings at various time points using just one plate. The AB is non-toxic, added at the beginning of the assay and cells followed over 24-30hrs. Cell numbers at the beginning of the assay should be titrated so that the reduction reaction is not too fast, but as a ball park figure 5000 cells / well of 96wp works well.

Alamar Blue is sold by a number of companies. We got it from AbD Serotec (Division of Morphosys).

PS. Word of warning:

I'm doing my toxicity assays in an hypoxic chamber with oxygen levels of 0.5% (which should model the in vivo environment under the skin). I find that I have to wait 20 mins after removing the plate from the hypoxic incubator to allow the reaction to equilibrate at the high 20% O2, before reading.

Gul
Gul's picture
MTT assay is a measure of

MTT assay is a measure of mitochondrial activity. My doubt is, can this method be applicable if cell toxicity has not affected the mitochondria. How sensitive is this method when a cell has entered the death pathway and still mitochondrial is functional?

Shubhangi
Shubhangi's picture
Hi

Hi
i had used all 3 -MTT,XTT and WST.
WST is most sensitive, less time required to get results but expensive.
XTT and WST liquid form and resulting formazan product is in soluble form.
MTT cheap-needs additional step of dissolving with DMSO or SDS
Using MTT gives good results. Instead of doubting reagent check if your cells are in healthy condition before seeding. it matters a lot.

Jith sn
Jith sn's picture
i was using MTT for my

i was using MTT for my cytokine proliferative assays, recently i started using CCK-8 but i find a lot of well to well variation in absorbance. Pls suggest me a solution

heehawmcduff
heehawmcduff's picture
You can also use MTS an an

You can also use MTS an an alternative to MTT - it is metabolized quicker than standard MTT and there is no need to solubilize with DMSO.
 

samm
samm's picture
Your other option is to use

Your other option is to use Alamar Blue or a flourescence based viabilty count - its great if yu are doing kinetics since the same replicate wells are reread over time.
As for your MTT, are you seeing variability in your cells alone replicates? Do these cells look healthy? Are you seeding too many cells/are there too many cells at the time of MTTaddition? How long is the MTT (can't be saturating)? How is the final dissolution step worked out?
 

santoshkumar
santoshkumar's picture
MTT assay is not useful for

MTT assay is not useful for those cells which are resistive of toxic effect of that particular drug or the drug is not showing significant changes in cell growth which may be due to inappropriate conditions or else.
If mitochondria is functional but cell has entered in apoptotic pathway it means, cell will stop its division and dies  after incubation for certain period which can easily monitored  by cell proliferation study of MTT assay .
 
 
Moderator note: MTT (and XTT,MTS) etc are NOT cell proliferation assays per se. They are bulk metabolic assays for normal cell function, and a quick way to check if a particular treatment has affected the redox health of a cell population.
Moderator note: Duplicate post deleted.

kranthi
kranthi's picture
Hi,

Hi,

I would like to know ,during MTT assay is it necessary to aspirate the medium before adding DMSO(stop mix)

heehawmcduff
heehawmcduff's picture
Hi

Hi

Generally, you leave the media containing the MTT suspension in the well as the DMSO acts to stop the reaction and solubilizes the formazan so you can measure the OD.

Tina84
Tina84's picture
hi,

hi,
i was doing the MTT assay and now i m trying to evaluate the data. Unfortunetly my gradient of the standartcurve is very low, something like 9x10^-5 (y=9*10^-5X+0,1), so it means the standartcurve is nearly plane and not rising. But in the graph it looks as if it`s growing fast. I dont really know if i m able to evaluate the data or if i have to change the cellnumber or something else? What are the normal data for a standartcurve you get out in a MTT assay?

naziasulthana
naziasulthana's picture
Dear cynthia

Dear cynthia

pls help me in performing MTT assy for GCSF using M NFS 60 cells

tolanvent
tolanvent's picture
I got your offer to help a

I got your offer to help a member wt d MTT assay. I am also working on antiproliferation properties of plant now and would need it, can you offer to us too? Email;   eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%61%74%6f%6c%61%6e%69%6f%40%72%75%6e%2e%65%64%75%2e%6e%67%22%3e%61%74%6f%6c%61%6e%69%6f%40%72%75%6e%2e%65%64%75%2e%6e%67%3c%2f%61%3e%27%29%3b'))

Arathi
Arathi's picture
Hi I was wondering to the

Hi I was wondering to the reply.. as in MTT assay, if u add DMSO it is required to aspirate media as due to presence of phenol red in the media which inhibits the correct OD readings!! I guess to non adherent cell this might differ..

vipanchal8
vipanchal8's picture
 Dear Vasipalli,

 Dear Vasipalli,

I am Vipul Panchal from India. i am a research student at IIAR Gandhinagar, Gujarat.

I have some problem with MTT assay.
My assay is working well but there is a lot of variation among replicates. The reason was while i was replacing media from 96 well plate some of the cells are getting disappeared. i took all possible precautions while changing media but each time i failed, that is some of the cells are getting expirate along with media. I am expirating media with micropipette.

To overcome this problem now i am thinking to perform MTT assay in 24 well plate wherein i don't have any problem of cell population change during media refreshment.  

The problem i have is of about references. All references contains that they have used 96 well plate for the assay. That means they are not having problems which i am facing. 

Since you have performed it successfully, it will be greatful if you can give some tips for media replacement in 96 well plate without disturbing cell population. Also i want to make sure that is it ok if i perform experiment in 24 well plate? I think there should be no problem.

Waiting for your help.

Norina
Norina's picture
Hi heehawmcduff ,

Hi heehawmcduff ,

I am really interested to learn about this other method of MTT assay. As from my reading, after 4h incubation with MTT reagent, some people used to remove the MTT medium before adding the DMSO, while the other would leave the MTT medium prior to DMSO addition.

Can this really be done (leaving the MTT+medium prior to DMSO addition)? Some says we have to aspirate the medium before adding DMSO in order to remove the unbound MTT that might interfere with the spectophotometric analysis (to avoid any nonspecific absorption and high signal to noise ratio).

I 've read some paper applying this method of leaving the MTT+medium before adding DMSO, but usually they leave it overnight before they measure the OD. Will the OD reading be any difference if we read the OD after the addition of DMSO.

Which one is the best option? What is the reason behind aspirating the MTT medium before adding the DMSO? And what is the other reason for leaving the media containing MTT suspension before DMSO addition.

I've always wanted to know this, but so far no one have the answer. I really hope you have the answer and can provide the references that I am looking for. Thank you very much for your time.  

DrakeICN
DrakeICN's picture
Adding to what Parvoman said,

Adding to what Parvoman said,

I have tried WST-1, neutral red, two other assays I do not remember and alamar blue.

Alamar blue is my favourite.

(I hope this does not count as advertise.)

DrakeICN
DrakeICN's picture
Oh and also, in a new post

Oh and also, in a new post since edit does not work for me for whatever reason, I use FACS on cells from the very same wells to which I have added alamar blue, and as far as I can tell, it is nicely washed away after two washes with PBS and does not conflict with PI. I mentioned this because alamar blue emits at 595 nm, the same as PI.

And also, becuase it someone else have another view of whether alamar blue conflicts with PI or not, it would help me improve my research.