Large variations in MTT - (Jan/18/2013 )

Control (Sample 1) 0.331 , 0.333 (Sample 2)0.457, 0.454 (Sample 3)0.450, 0.451
Treatment 1 (Sample 1) 0.390 , 0.374 (Sample 2)0.439, 0.458 (Sample 3)0.441, 0.444
Treatment 2 (Sample 1) 0.418 , 0.424 (Sample 2)0.446, 0.471 (Sample 3)0.406, 0.436
+ Control (Sample 1) 0.428 , 0.454 (Sample 2)0.429, 0.432 (Sample 3)0.441, 0.427


My cells are adherent cancer cells, seeded in 24-wells at 2500 cells/well and harvested on the 5th day of seeding.
Honesyly, I don't see much difference in cell density when view under microscope across all the wells.
Those data were obtained after incubation with MTT for about 2 hours, and solublised with DMSO for about 10 minutes with swirling.
The samples were then transfered to 96-wells plate for reading ( I pipet up and down)

But irrespective of that, I am expecting to get some of the treatment and + control to have about 1.5 fold increases over control... but the well-to-well variations of the same treatment set are quite big..I tried to avoid pipeting errors already..


Is there really serious problem with my pipeting? or could it be due to other factors? I have repeated the experiments several times and still don't get it work out.. What can I try to do next?
Please help... it's for my final year project which has very very tight schedule from now onwards... thx thx thx

Could be bubbles from the pipetting?

Volume plays a role in absorbance measurements - did you pre-wet the tip before pipetting the first wells?

A 1.5 fold difference should be visible with the naked eye - could you see something like this.

Cells settle really fast and trying to estimate if the wells have the same number is really hard by eye unless the difference is more than about 20%. It could easily be that you have fewer cells in the first wells of each replicate...

set up the experiment in a 96 well plate to reduce any pipetting error if possible,

and when removing mtt+media and before adding DMSO be very careful not to disturb the cell layer and alter results.

you can also try to add the DMSO and then watch it, help dissolve by pipetting, and then as soon as you think it's all dissolved transfer it to the 96 well plate - don't wait 10 minutes. the formazan should start dissolving right away and should be almost all dissolved within a minute I think; and perhaps spending too long in the DMSO is altering the results - I usually add DMSO, mix, and read, within 5 minutes total, and get very small errors.

are the control cells 100% confluent after 5 days or not? If not, perhaps you could plate all the cells at a higher density, so there are more of them at the end of the experiment for a greater total final reading.

it could be you are just using too few cells per well to see the difference between treatments. up the cell number to increase the final signal

Could it possibly have to do with the "edge effect" (increased evaporation in the outer wells of multi-well plates ?