Mystery - Cell Proliferation Assay - (Feb/13/2006 )
We are running cell proliferation to assays to determine the effects of several novel chemotherapeutic agents. Basically this involves taking our cells and exposing them to serial dilutions of a compounds for about 96 hrs.
Whilst most of our experiments perform admirably, producing wonderful sigmoidal curves that plateau at 100% at v. low concs (no difference in cell growth from control) and at 0% at high concs (complete inhibition of proliferation), we have noticed that on several occasions that the low conc plateau is at 30-60% of control levels. We have gone down as far as 10 fM which we calculate to be 40 molecules per cell.
We simply cannot understand why we cannot return to control levels with continuing dilutions.
I'm hoping someone else can shed light on this strange phenomenon. Perhaps you've seeen similar things yourself. I'm intrigued as to what is going on because it disobeys every pharmacological rule I was taught.
I'm not giving in to the homoeopathists just yet!
Thanks
well i see at time :
your treatment to deliver that drug is toxic by itself and you can't obtain more than the percentage of cells which survive that delivering procedure
drug is enough toxic to kill cells at very low concentration...
you have toxic agents that are not related to your drug and they do the killing job.
but i assume the last 2 possibilities would give little more survivying cells by dilution... but not ovious.
This simply cannot be the case because our control experiments (which are identical with the exception of drug content only) work fine and the cells survive and proliferate. The method of treatment and the vehicle do not contribute to the phenomenon.
We have also noticed that the use of different plastic plates and the practise of changing tips each time when creating the serial dilutions also affects the level of the plateau. No idea why. Any clues?
We have also noticed that the use of different plastic plates and the practise of changing tips each time when creating the serial dilutions also affects the level of the plateau. No idea why. Any clues?
Just a comment on plastic plates and cells.
We also noticed different cell behavior on different plates, either from different batches or different company.
Plates from the same company but different batch once had a circular growth like ripples and we cannot get confluent cell growth out of those plates.
Other plates from different company(which were bought out of price consideration), gave a wierd and abnormal cell morphology.
So, for the difference in the plates, I would say maybe the coating or treatment of the plates were different and might somehow causes differences in your cell morphology and cell function.
People seem to be getting a little confused. There doesn't appear to be any morphological or anti-proliferative effect produced by the plasticware we have tried. This is because the cells in the control wells grow perfectly fine. It's only when drug is added that the unusual behaviour appears. For some reason we can keep diluting to less that 40 molecules per cell and we still get up to 80% inhibition of growth. This simply isn't possible. At infinite dilution the results should mimick the control, but they do not.
It must be a drug plasticware issue or something even more odd. It almost seems as though it's not an active molecule that contributes to the effect. Hence why the results cannot be diluted away. Bizarre!
this is a wierd phenomenon...
can you do a shorter timecourse? maybe 40 molecules per cell is not limiting b/c it has so long to work i would try shorter incubation times???
may not be reasonable anyway just an idea hard to know without more info about molecules...
HTH
this is probably a really dumb question, but are you treating your 'negative controls' i.e. untreated cells with pure diluent? I was wondering if perhaps endotoxin (or something else) in the diluent could be the culprit?
I suppose this is reaching but it is the only thing I can think of to explain the results?