So, I am culturing human blood monocyte derived macrophages. I am hoping someone has tried doing the same.

I collect the blood in citrate anticoagulant, and then perform an autoMACS positive selection (usually 94% purity).
I seed the cells in endotoxin free RPMI complete medium (10% FCS, 1% Pen/strep, 1% L-Gln) and 500U/mL GM CSF at a concentration of 1×10^6 cells/mL in 96 well plates (2.5 × 10^5 cells/well). I replace the GM CSF every 48 hours by supplying 100uL of fresh medium containing the appropriate amounts of GMCSF.

I have a question regarding the morphology of the cells. They seem round and somewhat grainy in texture. How do you look for adherence and proper differentiation into macrophages just by confocal microscopy?

I notice that within 48 hours of cultures, colonies of proliferation form. Is this normal/desired?

I have cells which have been 3 weeks in culture, and their cell size is HUGE. Most wells which I have plated at a density described above now only have very few cells (maybe 1000 cells/well) but they are 10 times the size of my initially cultured cells. Are these still macrophages? If not, what could they be? They look like large live cells (shiny round and adherent).

Thanks!