Protocol to drive human monocyte-to-macrophage polarization in vitro using tumor conditioned media

STAR Protoc. 2022 Dec 16;3(4):101666. doi: 10.1016/j.xpro.2022.101666. Epub 2022 Sep 19.


Tumor-associated macrophages (TAMs) are key contributors to antitumor immunity. Here, we present a protocol to drive human monocyte-macrophage differentiation using tumor-derived conditioned media, followed by phenotypic and functional characterization of TAMs in vitro. We describe CD14+ cell isolation from healthy human blood, and detail the procedure to induce macrophage polarization. Finally, we outline morphological assessment of macrophages, and validation of their functional behaviors with a tumor cell killing assay. This translatable-based approach can be applied to different cancer cell types. For complete details on the use and execution of this protocol, please refer to Georgouli et al. (2019).

Keywords: Cancer; Cell culture; Cell differentiation; Cell isolation; Cell-based assays; Health sciences; Immunology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Separation
  • Culture Media, Conditioned / pharmacology
  • Humans
  • Macrophages* / metabolism
  • Monocytes*


  • Culture Media, Conditioned