Monocyte Polarization Assays

Monocytes are highly abundant circulatory effector immune cells and play a vital role in driving or resolving immune responses depending on their activation phenotype. In response to stimuli present in their local microenvironment or in circulation, monocytes modulate their gene expression towards either a pro-inflammatory (M1), an anti-inflammatory (M2) macrophages or dendritic cells (DCs).

Our assays provide a panel of monocyte polarization protocols of blood-derived monocytes or cell lines to achieve a stable, optimal and effective regimen for in vitro induction to drive antigen specific immune responses or immunosuppressive status. Our protocols allow the assessment of cell surface receptor expression, cytokine profiles, scavenging functions and ability to activate or suppress T-cell proliferation.

Human Macrophage Polarization 

Monocytes

M0

M1

M2

CD16-PE

CD80-PE-Cy7

CD206-BV-421

CD163-AF-647

CD14-FITC

Cell surface marker characterization of polarized macrophages

Isolated CD14+ cells by negative selection from human PBMC were polarized using indicated stimuli : 

M-CSF for M0 macrophages, IFNγ and LPS for M1 macrophages, IL-4, IL-13 and IL-10 for M2 macrophages. 

Phase contrast images of polarized macrophages were taken of the cells prior to flow cytometric analysis using antibodies against the mentioned cell surface molecules.

Basal cytokine production in polarized M0, M1 and M2 macrophages

The cell culture supernatants were analyzed for the production of key cytokines involved in the immune responses including:  IL-1β, IL-2, IL-4, IL-6,  IL-8, IL-10, IL-12 p70, GM-CSF, IFNγ, MCP-1 and TNFα using the Luminex platform.