monocyte isolation - why don't my monocytes adhere to the plastic??? (Nov/01/2005 )

Hello!
I am isolating monocytes from human peripheral blood, either by CD14+ MACS positive selection or by plating the whole PBMCs. Contrary to Paja who has difficulties to detach the cells from the plate, mine hardly stick to the plastic. After 1 or 2 hour incubation, when aspirating the suspension cells, only very few cells stay adherent to the plate. I also tried to use coated plates (gelatin, fibronectin), but I could'nt improve this. When I am stimulating the cells (IFNg, LPS...), then they begin to attach. Should "resting" monocytes isolated from blood naturally adhere to plastic? Thank you for your help!!!

hi,
even i observed that, my blood monocytes adhere to plastic very strongly.
researchers suggest that we should use cell culture flask for adhesion, because cells adhere very nicely. unfortunately i found that, it was very difficult to detach them from my culture flask and lost one experiment.
so in next round of experiment i used 6 cm dish with 2 million cell density for attachment, after one hour again i found same results like flask. my cells are pretty strong in adhesion. so i have got to trypsinize them for 10 minutes and even got to scrape them out in order to get my monocytes.

what i can suggest u is, i think the adhesion property may be variable between person to person.
so try using PBMCs from different donar. may be u will get expected results.

in terms of isolating pure monocyts i wud suggest u to follw the method mentioned in the paper, which sound logical and pretty simple (i have not tried this before).

http://memorias.ioc.fiocruz.br/952/3879.pdf

gud luck