MTT assay problems, while testing anticancer activity - (Mar/29/2009 )
Ponybn on Mar 29 2009, 08:45 AM said:
Are you sure it's ok to measure at 490nm?? This is 80nm away from the absorbance maximum of the purple formazan produced.
P
Penguin on Apr 3 2009, 09:00 PM said:
Ponybn on Mar 29 2009, 08:45 AM said:
Are you sure it's ok to measure at 490nm?? This is 80nm away from the absorbance maximum of the purple formazan produced.
P
hey penguin i am not sure whether we can do it at 490nm.. but i think it can it done.
DRT on Apr 3 2009, 05:49 AM said:
Would it be an option to forget about your samples for a couple of weeks and look to validate your protocol with a dose response curve for something like Triton X 100? You may find for instance that you need to increase, or acidify, the isopropanol; and possibly the cell seeding rate is too high if jurkat's grow well. Then you can include a ‘standard curve’ on each plate when you start assaying your extracts (this helps prove to supervisors everything is working).
Unfortunately the only way I can think of for getting rid of that sample background, apart from switching to another assay, is to include a 2nd or 3rd wash step to completely remove the extract.
Keep at it, it will work eventually.
The wash was done, i.e the supernatant was removed leaving the cell pellet and most of the sediments present in the extract also.. .well about washing with what should i wash? PBS? My tutor tells me that we cant wash the jurkat cells cause they are not adherent, i dont know if this is true.......
also are the formazan crystals formed from MTT soluble in PBS? what do u think from the results? does the extract have anti cancer activity? also i was told that the negative values show that cell proliferation is inhibited, is this true??
Thank you very much
Ponybn on Apr 4 2009, 02:03 AM said:
Sediments? I though your extract dissolved easily. I think you will always struggle with the MTT assay while you have extract sediment present. (unless it is possible to remove the solubilised formazan to a fresh reading plate at the last step while leaving all the cell/extract debris behind???).
Ponybn on Apr 4 2009, 02:03 AM said:
It is true they are not adherent, which is why you centrifuge before aspirating off the media. This is a step where care has to be taken or your cells will end up in the waste.
Ponybn on Apr 4 2009, 02:03 AM said:
No, that is what the isopropanol is for.
Thank you very much... i wil keep u posted of any upcomings... right now we are carrying out neutral red assay.. lets see how it comes up.. Thank you....... 
Hi.
I don't like the MTT assay. I had troubles too to get it running. Additionally, I think it is a bad measure of cell viability, since it greatly depends on the metabolic status of the cells. Try the Sulphorhodamine B assay. That's what I use to do assays like yours.
what is your extraction buffer, what its pH. Did you tried different concenration of extract diluted in plain culture medium.
Ponybn on Sun Mar 29 07:45:48 2009 said:
Hi... I am testing anti cancer activity of a plant extract on jurkat cell lines. I performed mtt assay and the od values were taken at 490nm instead of the 570 mentioned (because of lack of facilities ) THe OD values included negative values at high concentrations of my drug(20mg) .
I am just not able to know why this problem occurs, am a rookie who is just interested in cell culture and cancer biology.
The procedure i followed was,
1) plate the 96 wells, and incubate for 24hrs
2) Add my extract at different concentrations and incubate for 24hrs.
3) Centrifuge and remove the supernatant
4) add mtt and incubate for 4hrs and dissolve in isopropanol
5) Read at 490nm
if there is any errors please suggest, also my extract is very viscous and thick like a paste, i dissolved it in water and DMSO separately, filtered them through 0.45micron syringe filter and used it.
My tutor says its the problem with the extract.
PLEASE HELP
See doing centrifugation is bit risky as with the removal of media the formazan cytal can be removed. what we do is we add the 10ul of MTT(stock 5mg/ml) then keep it for 3 hr and then to the same we add solubilization buffer (20%SDS, 50%DMF).
further we take the OD at 570/650 after 1hr.
if you want the detailed protocol then let me know, i will mail you that.
good luck
Use WST-1 reagent from Roche if you can afford it:
http://www.roche-applied-science.com/pack-insert/1644807a.pdf
Your handling protocol has too many steps and that's where you introduce variation in the results.
Another more affordable option is resazurin (Tradename Alamar Blue, but buy in bulk from Sigma and prepare yourself):
http://en.wikipedia.org/wiki/Resazurin
Anton
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Sphere-forming assays have been widely used to retrospectively identify stem cells based on their reported capacity to evaluate self-renewal and differentiation at the single-cell level in vitro. The discovery of markers that allow the prospective isolation of stem cells and their progeny from their in vivo niche allows the functional properties of purified populations to be defined