Differentiation and Polarization of Monocyte-Derived Cells into Macrophage-Like Cells

Abstract

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection. J. Vis. Exp. (2020).

In this video, we demonstrate a procedure to induce the differentiation and polarization of peripheral blood-derived monocytes into M1 or M2 macrophages. Incubation with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and interferon‐gamma yields pro-inflammatory M1 macrophages, while incubation with macrophage colony-stimulating factor and interleukin-4 induces anti-inflammatory M2 macrophage production.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Differentiation and polarization of monocyte-derived cells

1. Use plastic adherence for isolation of monocytes. Briefly, seed freshly isolated peripheral blood mononuclear cells (PBMCs) in a 6-well culture plate at an appropriate concentration, e.g., 10 x 10⁶ PBMCs/well in 2 mL serum-free RPMI medium (Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine and 10 mM HEPES) ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Falcon 6-well Flat Bottom plates	Corning Life Sciences	353046
GM-CSF	Peprotech	300-03
IFN-γ	Peprotech	300-02
IL-4	Peprotech	200-04
L-Glutamine	GE Healthcare Life Sciences	SH30034.01
M-CSF	Peprotech	300-25
RPMI 1640	Life Technologies Corporation	SH30096.01
Fetal bovine serum (FBS)	Sigma-Aldrich	F7524
HEPES	GE Healthcare Life Sciences	SH30237.01
LPS (Escherichia coli O55:B5)	Sigma-Aldrich	L6529
Na-pyruvate	GE Healthcare Life Sciences	SH300239.01