20 replies to this topic

[cardosopedro]

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.

[THE ROCK]

what is your extraction buffer, what its pH. Did you tried different concenration of extract diluted in plain culture medium.

[scifistudent]

Ponybn, on 28 March 2009 - 11:45 PM, 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

[AntonAleraLabs]

Use WST-1 reagent from Roche if you can afford it:

http://www.roche-app...rt/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
Principal Consultant, Molecular and Cell Biology

Phone: (518) 334-0922
E-mail: antonb@aleralabs.com
Web: www.aleralabs.com

Alera Labs
P.O. Box 13019
Research Triangle Park, NC 27709

[Thomas Karmen DDS]

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

[nbio]

I am working withmacrophagesand am trying toassess the viabilityof theMTTmethod.The problem is thatafter the3 hoursincubation with theMTT, the mediumis alreadypurple,as if alreadyhad addedDMSO.Whatmight be happening?