Hi all. I have performed an MTT cell viability assay and the vehicle controls do not seem to be metabolizing the MTT reagent. I am basing this on the fact that I definitely see live adherent cells in the control wells yet I am seeing a very weak purple colour - looking almost the same in colour as the wells where I have treated my cells with anti-cancer compounds. Is it possible that the cells are not metabolizing the MTT reagent? I am working with MCF7 cells.
TIA.
Try to wait longer. I saw even an overnight incubation with Alamar Blue (an MTT analogue) as experiment for viability.
I utilized MTT assay for MCF-7 cells many times and it did work well. Are you sure that you dissolve MTT reagent in PBS. On the other hand, incubation time and concentration of reagent are also have an important role. If you give more detail about your procedure may be we would help you more.
FYI
Best,
Hi all. I am using the Roche Cell Proliferation Kit (MTT), so I don't make up any of my own solutions and I use it as per manufacturer's instructions. I add the labelling reagent (10ul per well in 96 well plate) and then 4 hours I add 100ul of the solubilizing agent (contains SDS) and then incubate at 37C O/N before I take my readings.
According to your procedure, it seems ok.
On the other hand you already use the kit which means you do not need to optimize something. 10ul solution 4 h incubation in 37 and then solubilizing with buffer 37 O/N that also what I follow for homemade way.
Are you sure that your kit have not expired already?
On the other hand may be the problem comes from the cell density. We seed 10,000 cell per well for this experiment. I hope you will solve your problem soon.
Best,
Thanks Yunus, I'm sure I will eventually figure this out! Aaah science!
I guess, in control, cells are stressed due to high density n may be because of that metabolically less active. try with low cell density
Hi Alexis, it's true that different cell lines can metabolize the MTT at very different rates. In my experience it seems that some lines only give a faint purple colour even if you leave them for a longer incubation, whereas others look strongly purple in under an hour! I did notice the protocol you describe has a final assay volume of 210 uL/well, which would make it difficult to detect a faint colour when it's that dilute. It sounds like you might benefit from a lower final volume which will increase the intensity of your signal.
There are a few ways of doing the MTT assay so here's what I suggest.
You can add the labelling reagent and do the 4 h incubation the same as the kit protocol, then remove the media. Wash with PBS (carefully), remove that, then add 50 uL/well of acidified isopropanol (or DMSO), agitate the plate gently for a few minutes to dissolve all the crystals, then read on the plate reader. If you have a faint colour you can reduce the 50 uL of solvent to 30 or 35 uL to increase the concentration and get a better signal. This way should work OK but if you still have problems then you can try the most reliable way of doing it (in my experience).
The other way of doing it is to remove the media and do a PBS wash (also remove this) before adding the MTT. Add 100 uL/well of the MTT at 0.5 mg/mL (you can make this up yourself, or if you dilute your premade labelling solution 1:10 in PBS this should be close enough). Incubate for as long as necessary for your cell line (up to 4 h but may be less; just keep track of it the first time). Remove the MTT solution, and blot the plate upside down on tissue to make sure all the solution is out of the wells (gently, to avoid the risk of detaching the cells). Then add the solvent, dissolve and read as above. I've found this final method is by far the most reliable and reproducible for the lines I've worked with but other people may have different experience; best advice is try it a few different ways and see what works best for your setup. Finally... Francis made a very good point so you may need to verify the compounds are killing the cells by other means. Good luck!
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