While making dilutions of a drug from stock to working, I have been using cell culture media (which is without serum). I used to make the dilutions fresh, but I cited a protocol which says they made dilutions in complete cell medium, which means they added serum to the drug dilutions. I don't think this should be done, because the drug may get entrapped in serum proteins and interact. Moreover, such dilutions cannot be stored at -20 C. Is there anyone who can enlighten me as to whether the addition of serum is appropriate or not?
I think either approach is OK, provided the same amount of serum is present in the final assay plate. Many drugs bind to serum and from a pharmacological perspective it is only the free drug concentration that will be active against your cells.
I think it would be better to use serum in your culture media, because serum protein binding of drugs occurs naturally in the body. This also helps to understand the accurate potency of drug on cells.
Avoid using serum as much as possible for preparing drug stocks, as eventually if your approach requires estimations of markers/enzymes/Abs etc., serum will interfere with all your readings..
Also drugs have different activity with or w/o FBS, so accordingly it should be chosen.
If your cells are growing in serum, then you are always going to have the issue of drug binding to serum proteins. If you are truly interested in eliminating this possiblity, then you need to have a serum-free cell culture system. Then you would make up your dosing solutions in the absence of serum.
Firstly serum wears off after sometime and thus it is not advisable to prepare drugs in serum for storage. Depending on the solubility of the drug, you should prepare and aliquots higher stock concentration and only dilute with the media in which the cells grow when you are about performing your assay. As said above, it does not make any difference whether the drugs are made with or without serum since in the end, the media of the cells if it contains serum will be much greater than your drug solution. Besides, the cells are supposed to survive naturally in the normal media first before the active ingredients in the drug kills them. Depriving them of a necessary requirement implies hampering their normal growth and might skewed your results.
Addition of serum will create bodily condition in-vitro (NOT 100%) but during the experiment (in standardization as well as sample analysis.), you do not have to forget about drug-protein binding.
I agree Patel. But you will like your experiments to be correlated with the natural. If the cells grow in a serum environment then it is essential that their conditions are maintained and the final serum concentration as well. So in the end you can say that your drug may have similar likely effect in nature
let me know the aim of the experiment with complete tentative protocol which will be followed by you.
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Alan Fairlamb...i agree either way is correct , but if i have to store the dilutions and then thaw at the time of use , so adding serum wont be a good idea
Harita Parikh....thats so very true, if i have to put up a dose response for the drugs with unknown function, then its necessary to first learn about the drug activity in pure form
Daniel Tagore ...Truly, it does not make any difference whether the drugs are made with or without serum since in the end, the media of the cells if it contains serum will be much greater than your drug solution. But again if we are storing them and reusing then the working concentration of the drug will be changed at different time points .
Urvesh Patel....Thanks a lot for sparing out time for my question. As you say adding serum will create almost a bodily environment in vitro, well, that is not significant for the experiment as the drugs are gonna be screened at primary level only. the only concern was if we can store those dilutions for a week and reuse them at different time-points or not , because the drugs will be degrading eventually .
I think it is better to use serum free medium, as the drug may interact with the serum proteins and this may affect the activity of the drug. For me I am using serum free medium.
The use of serum or not depends on the objective of your experiments. For drug discovery or relating in vivo to in vitro conditions, then serum should be present. Varying the per cent serum in the assay can be used to assess the extent of drug bound to serum proteins, if you don't have access to other methods for measuring drug binding to plasma proteins. The disavantage of including serum is the presence of nutrients that can by-pass the site of inhibition of a drug. FBS if stored frozen at -20 deg C is stable for years. We buy large lots of batch tested material to ensure reproducibility of our assays in the drug discovery unit at Dundee.
Thanx Alan, actually the study is about host-pathogen interaction, which includes various positives targets obtained from earlier work, so to avoid any kind of effect at the inhibition site , we chose not to use serum.
Neha, I think you have to investigate the role of serum enzymes, particularly proteases on the stability of the drug you are diluting. That would help you decide if you need serum or not in the stock solutions.
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