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

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).