I am currently performing hepatic immune cells isolation from high fat diet(HFD) mice. While separating immune cells from the liver extract using Optiprep and FACS buffer gradient , in case of HFD mice I am observing immune cells layer along with some junks which is clearly visible in microscope. Whereas separating immune cells from control diet mice (No high fat diet feeding), I am getting clear layer. Any suggestions as to what or why is the junk present in case of HFD mice and how can I remove the junk?
Is your 'junk' lipids? If so, make sure you add enough medium above the Optiprep layer to, hopefully, separate the cell layer from the lipids above the cells.
Also, you may want to do a wash before the density separation to remove cell debris etc, as well as a wash after the density separation.
what do you mean by immune cells?
Do you mean Lymphocyte infiltration?
My technique is easy and effective, sometimes my animals are also really fat.
First, you need to pass the liver by a 70um mesh, add enough PBS to wash 2 times (instead of a flick, take with a vacuum the liquid that is not the pellet) most of the lipids go. Then, instead of using Optiprep, I suggest using 40% Percoll with your pellet centrifuging 625g for 10 min.
I usually use 5mL per liver. You will just see the pellet at the bottom part with your mononuclear cells and all the other cells including lipidic and hepatic are going to go at the top as a monolayer that is easy to take out (anyway, be careful taking out the monolayer completely) and don't forget to wash the Percol and lyse erythrocytes to account cells.
Good luck and let me know if it works for you. :)
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