Monocyte-derived human macrophage culture - How much M-CSF should I add to enhance monocyte differentiation? (Dec/27/2007 )

Hello Everybody!

I'm currently working on human macrophages, derived from healthy donor whole blood, but unfortunately the harvesting of adherent cells through a multiple
Ca-Mg free PBS EDTA solution washing produces on the average 200.000 cells, while the desired quantity should be around 1 million.

In brief, the protocol I'm using is based on the isolation of PBMC out of 20 ml of whole blood with a Ficoll-Hypaque gradient, with a subsequent collection
of the cells, which are then incubated for 45' with 75 ng of M-CSF. After 3 days the colture mean is changed and another 75 ng of M-CSF is added. The
adherent cells are completely harvested, no significant residuals can be noticed with optical microscopy.

- Do you think that a total amount of 150 ng of M-CSF is not enough to allow a proper monocyte differentiation?
- Is it possible that 20 ml of whole blood are not enough to get the desired quantity of adherent macrophages?

Thank you very much for your help!

David

hi,
i was using 10-7 M or even less PMA/24 hours, all monocytes will stick efficiently.