Hi, everyone:
I'm looking for an efficient protocol for isolation of mononuclear cell from spleen and pleural effusion.
Ficoll-Hypaque density gradient centrifugation is a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample.I also found another protocol for "isolation of mouse mononuclear cells"(http://onlinelibrary.wiley.com/doi/10.1002/0471142735.im0301s39/full) .
1. I'm wondering if Ficoll can be used to isolate mononuclear cell from spleen and pleural effusion.
2. Is the "isolation of mouse mononuclear cells" better than Ficoll?
3. Are there other methods？
Please help me!
Hi,
Ficoll-Hypaque gradient it's fine. Be aware of the composition because it's dependant of the cell source specie. I think you can use the same method for blood and pleural effussions (and from elsewhere, the density of mononuclear cells should be the same).
Good luck
Hi,
I couldn't take a look at the protocol you posted, but i've used ficoll also for cell isolation from human tonsils, so there's no apparent reason it shouldn't work out with spleen. For the pleural effusion, you may need to use different ficoll:solvent ratio (i can't find my protocol, but for blood and tonsils we used different dilution with PBS).
I hope it helps!
Good luck! :)
As far as i remember, with the ficoll we used, there was a manual, explaining how much to dilute it, depending on what gradient you want to get, meaning what cell suspension you're going to use, which type of cells you want best to isolate and what part (relatively) of the overall suspension are they. It's a bit calculation, since you have to calculate the final density you want from the densities of the initial ficoll and the solvent you'll use. You need to include the centrifugation speed (fixed-angle rotor always) and maybe (i'm not sure about this one) the time also, since it's a combination, that leads to the specific density gradient.
http://www.sigmaaldrich.com/technical-documents/protocols/biology/ficoll-400.html
Here you can find some reference information, a bit simplified, without too much mathematics, so i hope it helps. I remember i used a protocol from a paper from the 1970s or smth for my case and it was still quite accurate. Some annoying thing is that even for such a standard chemicals like ficoll, different companies make more than one products, and of course publish different recommendations. This is the moment when you start cursing a bit, but if you don't find anything appropriate for pleural effusion you may need to adapt and optimize for your case...
Hope it helps a bit more now... :D
PS: You can try searching for your product first, companies usually provide detailed info about the densities etc. :)
Hi Hua,
here you can find different density gradients:
https://pluriselect.com/density-gradient-centrifugation.html
may be is worth trying.
Good luck.
Michael
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