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problem in human monocyte derived macrophage culture - macropahges do not stick to the plate (Jul/07/2011 )

I am working on human monocyte derived macropahge culture, i isolate PBMC from buffy coat, and obtaine monocyte by plastic adherence for overnight, after that, non adherent cells are wash and cells are scrapped in the presence of EDTA, then i culture monocyte at 0.5M/0.5ml RPMI 5% autologous serum/well in 24 well plate for 14 days, medium is changed every 3 days. Cells grow well and look like macrophage, but cells contiunue to detach when i change medium, even without changing medium and just by shaking the plate, cells detach and almost all the cells are gone. Isn't macropahge can stick to the bottom very tight? how to prevent cell detach during macropahge culture? i have try it for more than three months and the situation is still very worse. Can anyone help? Thanks very much


I think you checked already if your dishes are coated teh proper way ?