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?

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.

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

[quote=vasipalli]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.

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

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
[quote=RockyDoc][quote=vasipalli]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.[/quote]

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.

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

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 ?

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.

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?

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.

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

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.

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?