this might be a silly question but I have problems understanding better if a IC50 or EC50 (or any given compound concentration) is depending on the cell number used in the assay. I am not talking about e.g. confluency, pH changes, etc.etc. or any other cell culture effects that might arise due to cell layers growing over each other or senescence etc. Just from the principle.
Say I have compound X that I use in a 96-well format and in each well I have 1000 cells. I find out that I I have a receptor activation EC50 at 1 uM in a medium voume of 50 ul. If I now repeat the same test with 5000 cells per well, do I now get the same activation only when adding 5 uM compound X in 50 ul? The reason why I am asking is because I feel that the EC50 will be - within in the limits of experimental reproduciblity - again at 1uM. Or better always at 1uM. This because the EC50 is ultimately defined by affinity of 1 molecule compound to one receptor molecule.
Now someone is trying to convince me that it actually very much not only depends on the cell number but also the volume. I think this is wrong but I cannot really explain why it is wrong. For example:
- When I have an active compound at e.g. 10 uM this will be the needed concentration I will have to add independent of the medium volume. It is not the case that I would achieve the same effect by use double the medium volume of a 5uM concentration of the same commpound. The person argueing with me is saying that yes this would have the same effect on your cells as the cells now still "see" the same amount of compound in the well. Again, to me this feels wrong but I cannot really explain why
- Say a compound has an e.g. EC50 of 1 mM (so a quite inactive compound) in a standard assay with 10.000 cells per well with 100ul medium volume. If theoretically I now have 1 cell per well in 1ml volume, I would still need 1 mM of the compound to have the cell/Receptor "activated" and not only 10 nM compound concentration (because of 10.000 less cells and 10x more volume = 1mM/100.000=10nM)
Thanks, I agree...but why is this so? What is the response that will logically explain it why when I e.g. double the cell number I do not have to double the amount of medium or the concentration so that - per cell - the amount of compound per well is the same again? I know there must be some clearcut mathematical explanation, some eequlibrium equation that explains this but I do not know which one it is.
The crystal violet will bind equally well to DNA from adherent and non-adherent cells. It cannot differentiate between them per se. But through the staining protocol (e.g. the fact that one has to wash the crystal violet out at the end step) one washes away automatically the non-adherent cells.