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problems with MTT assay (some formazan crystals in the medium)


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#1 martha2

martha2

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Posted 02 May 2011 - 07:24 AM

Hi
I've had some problems with my MTT assay (some formazan crystals remain in the medium)
My protocol is the following
1) plate the 96 wells with 10,000 cell/well (final volume 100 l), incubate overnight
2) Add drug at different concentrations and incubate for 48hrs
Or incubate for 2h and then change the medium free from the drug and incubate for 48hrs
3) Add 10 l of 5mg/ml MTT dissolved in PBS (final concentration 0,5mg/ml) and incubate for 2hrs
4) Centrifuge at 2000 rpm for 7 minutes at room temp.
5) Remove supernatant and dissolve in DMSO.
6) Read at 570nm-690nm
690nm reference value
Im working with adherent cancer cell lines and using 96 well plate from TPP Techno Plastic Products 92096.

My problem occurs after step 3 and 4. I have some formazan crystal in the medium, so I thought by centrifuge the plate the formazan crystals would precipitate but the crystals remain in the surface of the medium.
So when I retrieve the medium before the addiction of DMSO I also retrieve some formazan crystals, so my results arent reliable.
Could somebody help me with this issue? Please
Another question: is my wavelength the most appropriated? I saw some protocols with other wavelength



#2 Hamed Karimian

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Posted 28 July 2012 - 06:43 AM

why do you centrifuge? there is no need of centrifigation, just remove carefully the media and add dmso...




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