Alamar Blue Assay - (Nov/03/2011 )

Hey Guys,

Does the alamar blue dye need to be added to the cells after particular time periods independently to determine cell viability or can it be added once and then checked over a certain amount of time (say 2 days)? The problem is that I'm not sure when my drug is supposed to actually affect the proliferation of my cells so I'm hoping to add the alamar blue as soon as I add the drug and then just check its fluorescence over time. I'm assuming that once the drug takes effect, the proliferation of the cells treated with the drug drops (the drug is supposed to be a growth inhibitor) so that in comparison to the control cells with no drug, I get a lower fluorescence. The issue is that just ran this experiment and it's been 24 hours but my fluorescence for both the treated and untreated cells have been the same. I'm hoping, though, that in another 24 hours, once the drug goes into effect (assuming it takes 48 hours to take into effect), I'll have higher flourescence levels in the control cells and lower levels in the treated cells. Will/Should this be the case?


Also, a lot of studies add the alamar blue at particular time points. For example, they'll treat with a drug, wait 24 hours, then add alamar blue and read flourescence only after 4 hours (and no time after). My question is--- is there anything wrong with adding the alamar blue at the same time as the drug? Shouldn't you get higher flourescence in the controls at some point regardless of when you add the drug (assuming the drug has anti-proliferative effects). Also, if I do decide to wait 24 hrs, then add the alamar blue, how long does it take for the alamar blue to actually reflect the number of viable cells that I have? Some studies check after one hour, others after 4. Any help would be greatly appreciate it!

Thanks so much~

I don't think adding Alamar blue (i.e. resazurin) at the same time as the drug will work. If the conversion of resazurin to resorufin (pink product) happens more quickly than the action of the drug, all the wells will turn pink and the fluorescence will be the same in all wells. The conversion of resazurin to resorufin is irreversible, so if resorufin is produced and then the cell dies, the fluorescence is still in the well.

The incubation time of Alamar blue can vary. I usually do 1-2 hrs. If you wait too long, all the resazurin in the well will be converted to resorufin and they will all be the same. If you don't wait long enough, there won't be good enough sensitivity. I would try a few time points on the same plate and just see what happens.

Thanks so much~ In a similar case though, if i'm adding a growth factor drug- --- shouldn't the fluorescence increase even more (become more pink) once the drug takes into effect, even if the alamar blue was added at the same time as the drug (assuming you never reach maximum fluorescence in treated and untreated wells) ? In other words, if at 24 hours after adding the drug and the alamar blue at the same time, I have all purplish/pink wells for both the treated and untreated wells (but not completely pink) because the drug hasn't yet taken effect, won't the cells with the growth factor proliferate at a much faster rate and cause a higher fluorescence (more pink) in comparison to the untreated cells in another 24 hours (assuming the drug takes into effect by then)?

Maybe I'm a bit confused but I thought that was the benefit of the alamar blue- that it allowed you to do a time course where you just add the alamar blue at a low enough cell density where you can check fluorescence over a period of time without exactly knowing when the drug goes into effect. Unless I misunderstood and what they meant by this ability to do a time course is that, as you said, you could check to see how long is required for incubation. If this is the case and I can't add the drug at the same time as the alamar blue, does that mean I would essentially need to know how long it takes for my drug takes into effect before adding the alamar blue?

Sorry for all the questions but I really appreciate your comments!