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Inconsistent MTT results


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#1 princesz

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Posted 15 November 2010 - 12:13 AM

Hello everyone.
I'm trying to figure out what went wrong with my MTT results.
I have done a few MTT tests to look for toxicity effect of edible bird's nest (EBN) on corneal keratocytes at different concentration.
Apparently the results were all not consistent; 3 of them having saw-tooth-like appearance and 1 appeared to show inhibitory-like effect.
My protocol :
1) cells seeding at 5000 cell/well in 96 well-plate in normal medium
2) cells incubation for 1 day to allow cell attachment; then normal medium is changed to medium+EBN
3) cells exposed to medium+EBN for 3 days (according to my study model)
4) 10ul MTT is added into cells+medium+EBN - leave for 4 hours in 37oC, 5% CO2 incubator
5) MTT+medium is sucked out from the wells - 200ul DMSO is incorporated to dissolve the formazan crystals. repeat pipetting up-and-down to allow complete solubilization.
6) Assay reading using ELISA reader at 570 wavelength

My questions are :
1) can pipetting error be the sole cause for my saw-tooth-like results?
2) I'm using pasteur pipette to suck the medium+MTT from the wells, so I think there's a high chance that 'some' of the purple formazan 'slipped' and being sucked out as well. can this be another factor contributing to my inconsistent result?
3) has any of u ever done MTT assay for edible bird's nest before?

I'm looking forward for comments/suggestions from u guys out there. Thanks!

#2 bob1

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Posted 15 November 2010 - 05:12 PM

What do you mean by "saw tooth"?


5000 cells/well may not be enough. I think 10,000 cells is the minimum for MTT activity measurement.

The formazan should not interfere with the MTT as it is measured at a different wavelength if I recall correctly.

#3 princesz

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Posted 23 November 2010 - 08:29 PM

saw-tooth = after a few concentrations, the graph peaks up, then on the next concentration the graph drops down significantly, and then it shoot up again on the next concentration...and this happens for 3-4 times in a few of my samples

#4 bob1

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Posted 24 November 2010 - 02:52 PM

Ok, sounds like you have a problem with mixing somewhere, which I would guess is during the cell seeding step - cells settle pretty fast, so you need to mix the cell suspension regularly when plating (I do it after every 8 wells if individual pipetting - approx every minute).

#5 princesz

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Posted 24 November 2010 - 07:41 PM

ok, noted! thanx alot bob..I owe u one ;p




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