Hi,
I have been trying to find the most appropriate protocol for myself to treat mouse splenocytes with fatty acids, but I am not sure what the most appropriate condition is for the experiment.
I have checked fatty acid levels in mouse plasma using mass spec and am trying to mimic the concentrations detected by mass spec. The plasma free fatty acids (FFA) was extracted using 90% methanol. The question is what is the best method to treat cells with different FAs if I want to mimic the plasma Fatty acid levels in culture?
I am considering between the two conditions below for my experiment
1. Complex FA to BSA and use FA-albumin complex to treat the splencoytes for the assay
2. Dissolve FA in an appropriate solvent (i.e. DMSO, ethanol, etc) to make FFA solution.
If any of you could give me great advice/explanation, that would be really appreciated. Thanks for your help!
Kind Regards,
Takumi
I have experience only with binding of fatty acids by several bacteria and you are interested in other possibility, it means to treat mouse splenocytes with fatty acids, but you are not sure what is the most appropriate. Igor Styriak
Hi Igor,
Yes, that's right. I am trying to treat mouse splenocytes with FA, but am considering what is the best way to mimic FA levels in culture. I guess I should use BSA-complexed FA as that seems the most common and closest to physiological condition as far as I know.
Thanks for your answer!
Takumi
You need to clarify:
1) to treat cells or to load cells with FA?
2) the purpose of this cell model
The Reason for clarification is: DMSO is not "neutral " for the cell plasma membrane and cell metabolism.
Otherwise, the both albumin and DMSO are suitable for reconstitution of FA in water-compatible solutions and delivery since your cell culture media most likely has FBS.
Hi Nikolai,
Thanks for your answer :) So I would like to:
1) treat cells with FA
2) the purpose of this is to check what biological effects free fatty acids have on mouse splenocytes. The readouts would be mitochondrial size and membrane potential.
I would like to mimic FA levels of plasma in cell culture and check what functional effects they would have on metabolism and function of mouse splenocytes. I would like to minimise the FBS as well since serum would contain FAs as well. But considering free fatty acids are transported on albumin in circulation, would you suggest I complex with BSA?
Thanks for your help!
Kind Regards,
Takumi
the effects of fatty acids on cell membranes and metabolism will be different according to the presence or absence of calf serum in your culture medium. If it is possible for you, I will work on serum free cultures to avoid interferences with fatty acids bound to serum proteins. One way to add the fatty acids in the cultures would be to dissolve the molecules in ethanol, put a known volume of the solution on the bottom of your culture flasks, evaporate the solvent and then add your cultures in the flask and let them incorporate the substrate. You may thus compare the reaction of your cells in the presence of various amounts of the fatty acid tested.
Good luck
Hi Dominique,
Thank you for your advice. The protocol sounds good, and I would like to have a go :) On the note, two of the FAs I would like to use are oleic acid and linoleic acid, I wonder if the evaporation of the solvent can affect their chemical structures (i.e. they may be oxidised and lose double bond)?
Could you please advise if you have any ideas?
Thank you so much!
Kind Regards,
Takumi
to avoid oxidation you may evaporate the solvent under nitrogen gas flow
Best regards
Dominique
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