A simple and effective method for the isolation and culture of human monocytes from small volumes of peripheral blood.

Innate immune cell defects contribute to severe autoimmunity and the pathogenesis of inflammatory disease. Monocyte-derived macrophages typically retain disease related signatures and represent an excellent in vitro model to uncover and validate mechanisms contributing to specific pathological states. Monocyte isolation procedures vary widely in terms of purity, yield, cost, degree of technical difficulty and volume of peripheral blood needed. This paper outlines a novel isolation method that yields monocytes through density gradient centrifugation (Ficoll® and hyperosmotic Percoll®). The protocol has been optimised for small volumes of blood (42 ml) and is simple, reproducible and inexpensive compared to other methods. Monocyte recovery is 70% (relative to monocyte numbers within the buffy coat) and the highly functional macrophages produced are characterised by excellent purity (98.6 ± 0.6%) and intact activation and phagocytic capacities. The method is well suited to investigations involving patient populations where a particular subset of immune cells is known to contribute to the pathogenesis of a specific disease or is aberrant as a consequence of that disease.