In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Abstract

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)

This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF).

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. Isolation of primary human monocytes by immunomagnetic negative selection

1. Collect 40 mL of fresh, human whole blood (from either an HIV⁺ patient or healthy control) in four 10 mL ethylenediaminetetraacetic acid (EDTA) vacuum tubes (10 mL of blood per tube). Using sterile techniques under a biosafety cabinet, transfer all 40 mL of blood into one 50 mL conical propylene tube.<....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5M EDTA	Invitrogen	AM9260G
BD Vacutainer Plastic Blood Collection Tubes with K2EDTA	BD Biosciences	366643
Easy 50 EasySep Magnet	StemCell Technologies	18002
Easy Sep Direct Human Monocyte Isolation Kit	StemCell Technologies	19669
Fetal Bovin Serum Defined Heat Inactivated	Hyclone	SH30070.03HI
Nunc 35mm Cell Culture Dish	Thermo Scientific	150318
PBS	Gibco	20012027
Penicillin-Streptomycin	Gibco	15140122
Recombinant Human GM-CSF	R&D Systems	215-GM-050
Recombinant Human M-CSF	R&D Systems	216-MC-025
RPMI 1640 with L-Glutamine	Corning	10040CVMP