Introduction

FIG. 1.  Human blood monocytes in cell culture medium under low magnification using phase-contrast microscopy (40x objective). Note the refractive appearance, pseudopods, and irregular cell morphology of unstained monocytes in culture.   

Methods

Using magnetic cell separation, blood monocytes were purified from human peripheral blood using an AutoMACS (Miltenyi Biotec) magnetic cell separation instrument. The monocytes are labeled with a monocyte-specific monoclonal antibody conjugated with a magnetic nanoparticle. The cell mixture is subjected to a gradient magnetic field in a column; the labeled cells are retained and the unlabeled cells are eluted from the column first.  Finally the labeled monocytes are eluted from the column. In this experiment, positive selection was performed using anti-CD14 antibody to purify monocytes expressing the cell surface antigen marker CD14 (cluster of differentiation 14). Purified monocytes were cultivated at 37^oC under 5% CO₂ in Dulbecco minimal essential cell culture medium containing 5% calf serum.

Discussion

Monocytes are immature phagocytic cells found in the blood that have the ability to internalize microbes and particulate matter. Monocytes belong to the  innate immune (nonspecific) system and mature into macrophages after migrating into tissues. Macrophages are migratory cells found in most tissues of the body and are essential for both innate and adaptive immune defenses (specific), where they engulf and digest cellular debris and recognize pathogens by a series of toll-like receptors to activate the immune system to respond to various pathogens.

Purification and cultivation of primary human monocytes as an in vitro culture system is essential for investigating host-pathogen interactions.  Monocytes and macrophages are hosts for a variety of pathogens with potential to be used as biological weapons. For example, Bacillus anthracis (1), Francisella tularensis (3), Yersinia pestis (4), and Coxiella burnetii(6) all interfere with normal macrophage functions, which result in enhanced virulence of these select agents (1). Monocytes and macrophages also serve as hosts for several emerging infectious diseases including tuberculosis (2) and Dengue virus (5).  

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