I use monocytes that were isolated from PBMCs via adherence to tissue cultured plate. We differentiate them to macrophages via adding 10 ng/mL of MCSF in RPMI +10%FBS + 1%P/S on the 2nd and 6th day of differentiation. We confirm that that monocytes have become macrophages via the "sunny side up egg" appearance of the majority of cells by day 7. We usually get at least 80% confluence of the cells and at least 75% of the cells have the egg appearance. Then we start our lipid loading experiments, one day with 10% lipoprotein deficient serum, then in aggregated lipoprotein solution for 24 hours, and run them on flow cytometry.

The differentiation takes 7 days and the experiment takes 3 days. We've also started using both fresh and frozen PBMCs, I did not notice a difference between the fresh and frozen PBMCs, except for the slower differentiation of the frozen cells.

a). I've confirmed the egg appearance of the cells, however, when I evaluated their lipid content (using BODIPY 493/503), the macrophages seem to not have taken up lipids. Which is surprising as they should be actively phagocytic.

b) is confirmation of the differentiation (via appearance) sufficient? or could they look like macrophages but do not have the phagocytic capacity?

c) In another set of experiment, the cells did not have the egg appearance on the 6th day but look like larger monocytes. Should I look into the cells or the MCSF?

d) Is there a difference in frozen and fresh macrophages in terms of lipid uptake?

Thanks in advance for your help!