I´ve been having a lot of contamination problems when carrying out assays at "high" enzalutamide concentrations (>100uM) in cell culture media. I´ve carried out different tests to determine which is the source of contamination and it always points towards the drug.
I´ve tried enzalutamide from two different companies (sigma and cayman) both of them specify that they can´t ensure that the lyophilized drug is sterile, so they recommend to filter the solution although the drug is dissolved and DMSO which is supposed to be "sterile".
When carrying out the experiments using the drug dissolved in DMSO without filtering it. I always got bacterial growth at high drug concentrations (>100uM), but at lower concentrations i won´t get bacterial growth, even though all the treatments are prepared from the same enzalutamide stock solution (this happened with both companies).
When filtering the solution (Enzalutamide in DMSO at 100uM diluted in cell culture media at a final concentration of 1uM), i get rid of the contamination problems, but i always notice a severe decrease in drug activity (close to negative control). Could this be due to loss of enzalutamide during the filtration process? How else could i sterilize these solutions?
The other option is to use cell culture media with P/S, but it seems that this doesn´t get rid of bacterial growth, at least with Sigma´s enzlautamide and im not sure if antiobiotics could interfere with my results.
Parallel to this experiments i always make a "copy" of these with bicalutamide (also an antiandrogen), and everything goes as expected.
@Rachel Gad
The last vial that i recently obtained (cayman chemicals) had 5mg of drug and was resuspended in 100ul of DMSO inside a hood and always under sterile conditions. No filtration was made. The problem persists.
The issue that i have with what you propose is that i need high drug concentrations in my experiments (between 300 and 1uM). If i prepare a stock solution at a concentration of 1mM (DMSO 100%) at the end, the final working concentration of DMSO in media will be toxic.
The other problem that i have is the volume of stock solution (100ul), if i try to filter that amount of volume i will loss a significant portion of the solution.
I normally filter a "mid-way" solution which is prepared by diluting stock solution (DMSO 100%) in culture media at a final concentration of 1mM. The problem with this method is that i have noticed a loss of activity, which led me to think if some drug might get "trapped" on the filter.
Currently, my stock solution is at 100mM (enza) in DMSO (100%), 100ul in total. As of right now i don´t have filters suitable for these amounts. I could try preparing a diluted solution using DMSO 100% to obtain a final enza concentration of 10mM.
The problem is that if i use this concentration (10mM, DMSO 100%)and my treatments need to be at 300, 200 and 100 uM, i´ll need to dilute 3, 2 and 1 ul of the 10mM enza solution into a final volume of 100ul and the final DMSO percentage in each treatment will be 3, 2 and 1%, which i have already test and it ends up killing the cells in the DMSO control. "Safe" DMSO concentrations are below 0.2%, that´s why i can´t use a stock concentration of 10mM. I could try preparing even more concentrated stock solutions, but I´ll be getting close to the max solubility threshold.
"and try diluting the recommended concentration in serum free medium", does the serum in media interferes with the filtration process? If i do this i guess that i will have to use small amounts of this serum-free media with my treatment and take it to a final volume of 100ul (well volume) using media with serum, right? I normally culture my cells with FBS.
All of my calculations to prepare my stock solutions from scratch were made using the "calculator" and considering the dilution table from selleckchem´s website. Also making manual calculations to double check the amounts.
Something else that i haven´t done, or consider, from what you are telling me is using a 0.45uM filter. The filters that I have been using (since those where the ones at my disposal during the time being) have a pore size of 0.2uM. Do you think this could promote the "trapping" of enzalutamide in the membrane?
The first option that you show is how i normally prepare my treatments.
From what i have read, my only option will be sticking with filtration, although i won´t be able to filter the DMSO solution. I´ll need to prepare the 1mM media + enza solution in order to have enough volume and the lowest DMSO final concentration possible.
The second option will give me toxic DMSO levels.
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