How do I prevent precipitate formation in case of challenging cells with palmitic acid?
I guess you can use DMSO if you are working with low conc. of palmitic acid, but you would need to to a DMSO only control. Cells have different tolerance for DMSO so you would have to establish a limit for your cells
Serum albumin is the most abundant protein in plasma (and in tissue culture fetal bovine serum) and doesn't enter cells. It is also the major fatty acid carrier protein in vivo so it is more physiological.
Hi, there are 2 ways I know of: 1) sonicate the palmitic acid-aqueous solution into micelles and add them or 2) saturate fatty acid free bovine serum albumin (BSA) with palmitic acid micelles.
Thank you Steingrimur. I wanted to ask isn't it possible in DMSO?
And since i will be doing protein work doesn't BSA makes difference to the total protein cancentration?
I guess you can use DMSO if you are working with low conc. of palmitic acid, but you would need to to a DMSO only control. Cells have different tolerance for DMSO so you would have to establish a limit for your cells
Serum albumin is the most abundant protein in plasma (and in tissue culture fetal bovine serum) and doesn't enter cells. It is also the major fatty acid carrier protein in vivo so it is more physiological.
Steingrimur, thanks again. I will be using 0.2mM palmitic acid to challenge the huh-7 cells.
I do use oleic acid (0.1mM to 0.3mM), every now and then, to get significant numbers of lipid droplets inside the cells. For that I generally use to make 20-30 mM Oleic Acid stock precomplexed with defatted BSA. I generally add Oleic Acid in 2.5 mM BSA (fatty acid free, cell culture tested BSA in Ca/Mg free 1X PBS) and sonicate to emulsify it prperly.
I think you can do the same with palmitic acid also before challenging your cells.
Good luck!
Hi everybody, i'm also curious about the use of oleic acid for cell treatment. Do you just diluite your oleic acid in 2.5mM BSA (fatty acid free, cell culture tested BSA in Ca/Mg free 1X PBS) and sonicate, and that's it?
Hi Sweta and Steingrimur, I am wondering if this sonicate method also works for unsaturated fatty acid (e.g. docosahexaenoic acid)? what device should I use and how long shall I sonicate the fatty acid-aqueous solution?
I am currently sonicating the solutions at 40kHz for 1 min right before I add them, don't know if this is the optimal/standard procedure. I do have noticed robust changes in the cell challenge results compared to un-sonicated solutions.
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