I use CD14 micro-beads and MAC cell separator for the separation. Do you think Dynabeads rather than Microbead will yield more Monocyte.

We use Ficoll gradients to prepare PBMCs (http://www.stemcell.com/en/Products/All-Products/FicollPaque-PLUS.aspx) and then select monocytes via CD14 beads from Milteny (https://www.miltenyibiotec.com/en/Products-and-Services/MACS-Cell-Separation/Cell-separation-reagents/Monocytes-and-macrophages/CD14-MicroBeads-human.aspx).

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Will you be adding any cytokines to your purified monocyte culture (like M-CSF for macrophage differentiation)? My pure population of monocytes starts to die off the very next day after I plate them (with or without cytokines, although with IL4 and GMCSF [for dendritic cell differentiation] it was not as bad as without any cytokines). I think they need other cells in culture to stay healthy. If you don't need very pure population, you might try plastic adherence. This will result with lower purity of monocyte culture due to remaining T cells (and possibly other cells), you might be able to keep them alive and healthy for a bit longer. Good luck!

Hi Nasrin,

Thanks for your suggestion. I use Ficoll too to prepare the PBMC and then CD14 microbead. But don't know why, the yield is not good. I get 0.4%-4% monocyte out of 10*10E6 PBMC. I think thats too low yield. I need very pure population and that's why I am concerned.

Nasrin ghassemi barghi Neither one of the links that you supplied worked when I tried them. I tried to copy the link to my browser and that did not work either??

Hi Nasrin,

Would you mind to share your Monocyte Isolation from PBMC protocol, please? I tried to go through the link but it didn't work. I will appreciate your response.

Thank you.