Phenotypic Characterization of Human M1 and M2a Macrophages Cultured with Different Serums and in the Presence or Absence of Polarizing Cytokines


Macrophages are ubiquitously distributed throughout the body and perform many functions including inflammatory responses against pathogens by classically activated M1 macrophages and the regulation of wound healing and tissue remodeling by antiinflammatory alternatively activated M2 macrophages. The responsibility for these pleiotropic functions lies in the expression of surface receptors unique to each subset.

As new techniques such as RNA-Seq have become available, it is becoming clear that a more robust analysis of macrophage surface marker expression is needed to understand the spectrum of macrophage activation. Concomitantly, it is also necessary to understand how the in vitro culture conditions used to generate macrophages affect the expression of various surface markers. More recently, expanded sets of M1 and M2a markers have been described for macrophages cultured in media containing fetal bovine serum (FBS) and polarized with IFN-gamma and LPS (M1) or IL-4 and IL-13 (M2a).1 Still, it remains unclear how these phenotypes compare to macrophages grown in media containing human serum or serum-free media and whether polarizing stimuli are necessary for the expression of various surface markers.2,3

Here we begin to delineate how the culture conditions used to generate human M1 and M2a macrophage subsets affect the expression of multiple surface markers. We directly compared the expression of fifty different surface markers on M1 and M2a macrophages cultured under various conditions. Of those fifty, we show how the presence of FBS, human AB serum or serum-free media and polarizing cytokines affected the expression of twenty of these markers such as CD200 R1 and CD32. Moreover, the inclusion of polarizing stimuli was critical to the expression of several of these markers such as CD38 and SLAMF7. These results significantly expand our knowledge of the phenotypic differences between human M1 and M2a macrophages as well as demonstrate the importance of culture conditions in generating these phenotypes.


  1. Beyer, M. et al. (2012) PLoS One 7:e45466.
  2. Rey-Giraud, F. et al. (2012) PLoS One 7:e42656.
  3. Vogel, D.Y. et al. (2014) Immunobiology 219:695.