Isolation of monocytes by adhesion

[Bijou]

Hi,
I would appreciate advice on the following:

I am having trouble isolating monocytes by adhesion, the method I use is incubating the mononuclear cell fraction ( monocytes + lymphocytes) for either 4 hours in serum free, high glucose DMEM or overnight in RPMI +10% FBS. The washing twice with medium (i.e. pipetting out medium, pipetting new medium on the bottom of the flask in a "gentle flushing manner"). The scraping the cells off and resuspending them in medium.
It's not working very well and at the moment I don't have the money to buy a magentic separation kit not use percoll based methods.

Please advice! I am quite desperate.

Thanks in advance.

[procopio f.a.]

this method is cheap but far long to be perfect. In effect you will get a lot of contamination by T cells. Beads against cd14 is for sure the best way to go.
in alternative you can let the cell adhere for 1 hour only, keep the plate in ace for 15 min (the t will detach faster) wash very well with cold pbs, and just after try to scrap your monocites. But like i said it s difficult you will get great results.
procopio fa

[Aarfy]

I'm not sure I understand the problem. Is it that you're not getting adhesion? Or are too many lymphocytes adhering after washing and you're not getting a very high percent of monocytes?

The method we've typically used, was an overnight incubation in RPMI+10% FPS+Benzonase (or DNAse, this is to prevent stickiness by DNA if the cells are damaged, not really necessary for fresh cells but definitely suggest adding if getting cells from freezer). Next day, remove the supernatant and wash 2-3 times with ice-cold 1xPBS. Typically what remained was a good population of monocytes. Our purpose was to get dendritic cells so we would changed the media conditions and then harvest by scraping.