Hello everybody,
I have some issues with my pharmacology study. I have different EC50 for one compound (one of my cyclic nucleotides) between frozen and fresh cells... It's like it's more active in the frozen cells (?) than the fresh cell (read out= reporter gene expression/secretion of the SEAP in the media, and I use QuantiBlue to detect the SEAP). It's like I have a shift of my curve in higher concentration for the fresh cells (can't reach the plateau for the highest concentrations)>
For one of the other cyclic nucleotide no difference at all between frozen/fresh cells... So do you have an idea about what does that mean? Have you already noticed this type of issue?
thanks a lot in advance,
Alice
Dear Alice,
Interesting observation. Most probably it is our so called biologically '' inert'' DMSO! If you have used DMSO as a cryoprotectant for freezing your cells and you are not sure that some of it might have reached in your assay mix then the observed effect is mostly because of DMSO. We have observed some very interesting effects of DMSO in certain pharmacological assays and it actually shifted the DRC of one of our compound towards left. Hope it helps!
Best Nand
Dear Alice,
Freezing is an important distress for the cells and it can change their metabolism and could also affect gene expression patterns. In my experience, cells respond very different when frozen compared to fresh. Membranes are one of the most frequent and important damage, hence it compromise the cell compartimentalization and the overall response to any external compound they are exposed to. It cound also make intracellular proteins to release to the medium, even if your protein of interest is a secreted one, you might find higher amounts because of this phenomenon.
Also cell viability is strongly compromised but, depending on the cell type, you may have a non-negligible amunt of "diying" cells which mask the effect of create artifacts.
Hope my thoughts could be helpful. Good luck and best regards.
Thanks a lot for all your answers. As you said Manuel Lee, I suspected a difference in cell membrane permeability/protein expression/compartimentalization... The thing is is more convenient to work with frozen cells in HTS... Fortunately, I have an other compound that gives me the same pharmacology :the EC50 is the same even if the cells are frozen/rested/fresh (just the DO is a bit lower in the frozen cells but increases in rested cells, they need some time to have their metabolism restored).
Thanks a lot again .
Have a nice day,
Alice
I would agree completely with my colleagues that the freezing of cells is not a natural process, and in some way change the metabolism of cells as opposed to fresh cells where everything is natural, I think they did very well specifically and clearly documented!
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