I've been working with the MDA-MB 231, MDA-MB 157 and MCF7 breast cancer cells. With each cell line except for the 157s I saw a consistent trend in the decrease in % viability with the MTT assay. Each replication of the experiments produced similar results, my n was 32. In our lab it is standard to do different experiments that test almost the same thing but at different parameters. I decided to do the Annexin V apoptosis assay. I expected to see a similar trend as compared to the MTT assays, however in spite of seeing increases in apoptotic cells and necrotic cells with the incorporation of my compounds, the trend is different. Where in the MTT the lower concentrations of the compounds in combination showed greater efficacy in regard to decreases in cellular viability, the data from my apoptosis assay is showing greater apoptosis and necrosis at the higher combinations. Is this unusual and should I keep repeating the apoptosis assay, or is there an explanation for what I am seeing? I figured it could be that cells are not growing as much with the "more effective" combination thus not as many cells are undergoing apoptosis due to the fact that the cells aren't there. The MTT assay measures the amount of viable cells whereas the Apoptosis assay only measures the cells undergoing apoptosis. I'm also doing colony forming assays and so far these show that the colony forming potential decreases with my treatments. I also took photographs of the cells after treatment and noticed that they appear to be less confluent at the lower combination which is consistent with the MTT assay. I believe that cells are not only dying but their continued growth and proliferation is being inhibited. Would this be a sufficient explanation?
At low dose it induces cell cycle arrest and at high dose it induces apoptosis. Do a PI-FACS that will tell the quantitative story of both cell cycle arrest and DNA fragmentation (apoptosis) in one shot. If you see minor shift of G1 like peak towards DNA fragmentation (sub-G1) side, that indicates necrosis; whereas a major shift is apoptosis. Good Luck.
I share Goodwin's opinion.
You did not clearly indicated at what timing you performed the MTT experiments versus the FACS ones? Different timing with different tests can provide you with different responses.
You could also maybe sharing with us morphological images at both low and high magnification to see the morphological characteristics of your cells under the various experimental conditions you used.
Best regards
Robert
Dear Kendra,
it is important to take into consideration that MTT assay does not determinate apoptosis or "real toxicity" : it is a colorimetric assay for measuring the mitochondrial reductases activity in living cells. It will give you a value corresponding to cell activity.
Of course you can correlate cell activity with viability but a correlation with apoptosis is more tricky. I will try to explain my opinion:
day 1: stimulation
- (A) 100.000 cells are kept untreated,
- (B) 100.000 cells are serum-starved (we suppose they will not grow)
- (C) 100.000 cells are in contact with apoptotic agent.
day 2: measurments
- (A) untreteated cells have proliferated (now 200.000 cells),
- (B) starved cells did not grow (now 100.000 cells) ,
- (C) and 50% cells died under apoptotic agent (now 50.000 cells).
let's do a MTT vs annexcin V-PI apoptosis assay comparison:
- (A): untreated cells become reference for MTT: 100% ; annV-PI : 100 % viability
- (B): starved cells: MTT= 50% reference; annV-PI: 100% viability
- (C): apoptotic cells: MTT = 25% ref; annV-PI: 25% viability
In these cases, MTT does not always correlate with annV-PI staining and cells that do not proliferate can have a low MTT but not be apoptotic.
I hope this can be of help; in case you would like to try an alternative to your MTT you can find reference of ours; you can contact me directly at [email protected] for more information.
best regards,
Cedric
http://www.ozbiosciences.com/assay-kits/51-mtt-cell-proliferation-assay-kit.html
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