macrophages - isolating monocyte-derived macropahes (Dec/07/2005 )

Hi everyone, i am trying to isolate macrophages from monocytes obtained from blood using buffey coas method and planning to select macrophages using CD14 beads and then grow them with rM-CSF. Has anyone tied this method or have any alternate suggestions to this.

Thankyou for your help.

Pubgirl

Definitely it is possible. You can isolate PBMC, treat them with CD14 Microbeads (Miltenyi's work well), and culture your monocytes for in vitro differentiation. In vitro, you can use either mCSF or PMA as differentiation factors. Be sure to obtain a good number of monocytes, since in vitro, these will not reproduce. (M-CSF and PMA are differentation factors, not growth factors.)You can treat monocytes for 2 days with mscf and then incubate them for up to 12 days, feeding every 2-3 days.

Hope this helps,

Good luck!!