We always isolated PBMCs successfully from mouse blood with Ficoll and density centrifugation (700g, 25 minutes). However, in the last experiments we observed an incomplete separation of granulocytes from the PBMC layer and therefore the PBMCs were impure. We don't observe this problem with human blood, we isolate PBMCs from human blood several times a day and the separation of PBMCs and granulocytes/erythrocytes is perfect.
What we already tried:
- Second Ficoll step. We successfully removed the granulocytes but monocytes don't like prolong exposure to Ficoll and they die.
- We changed the ratio of Ficoll to blood but this doesn't led to a better separation.
Any ideas what might be the problem?
Dear Manuela,
It may simply be a problem of density. The density of Ficoll-Paque is 1.077 g/ml. Cedarlane sells a density gradient suited for mouse PBMC. It is called Lympholyte-M and the density is 1.0875 g/ml. I think it is worth trying it.
Best,
Stéphane
Ficoll gradient has two basic rules: temperature and dilution of blood.
For the first, is important to have the correct temperature in both liquids, blood and ficoll when you prepare them: 18ºC.
For the second, a minimun of 1:2 deplaquetized blood dilution with RPMI or PBS may be neccesary and a ratio of 4:5 of ficoll-blood for the run are required. To improve the purity try to dilute the blood much more: 1:3 or 1:4 with tempered RPMI.
If you have problems with granulocytes it also may be due to a termic shock in mixing components: ficoll, RPMI or blood at very diferent temperatures.
Hi, there are factors you must put into consideration to ensure standard. You have to consider the density of the Ficoll, temperature and dilution of the samples.
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