Many Thanks, Andre

Dear Bing Hou

First of all it is not possible to proliferate the monocytes in vitro it can only be differentiate into macropages in presence of MCSF in 3-6 days.

The number of monocytes in blood is very low (0.2–1.0×106/ml of blood). So you should seed more number of PBMC's to increase the total number the monocytes

The most important thing is, culture your cells in RPMI 1640 media without FBS for at least 4 hours for better attachment of monocytes.

All the best

I agree with Abhishek Vats, in ours hands the monocytes don't proliferate. We obtain up to 5-10 x108 monocytes from  buffy coat.

Good luck

Depending on your type of selection/negative selection you can also estimate that about ~18-20% of your PBMC will be CD14+ and differentiate into macrophages. You may want to scale down your experiments or use an alternative source for cells.

Dear Bing Ho

It is possible, but only in one direction, culture of monocytes to macrophages and then to dendritic cells in the presence of IL-4 and GM-CSF. Due to the fact that we need to have a large volume of blood, we use the buffy coats.

I am sorry to say that human monocytes do not proliferate in vitro. So I agree with Matthew that you should either find another monocyte source or downscale your experiments. You could isolate all monocytes and not only the CD14+ ones.

Many thanks everybody!

I am confused...This paper say monocytes can be subcultured...

http://www.izs.it/vet_italiana/2011/47_2/139.htm

http://www.izs.it/vet_italiana/2011/47_2/139.pdf

Hi Mingkuan,

Our lab (Hedrick lab at LJI is very experienced in working with mouse and human monocytes) is trying to culture the individual human monocyte subsets. We find so far they start differentiating or start dying after only 2 hours in culture. We check this every time by Flow cytometry. We have also tried RPMI 10%FCS like in the paper you mentioned.