title: Impact of human granulocyte and monocyte isolation procedures on functional studies.
abstract: One of the first lines of defense against infection is the activation of the innate immune system. It is becoming clear that autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, may be caused by disturbed innate immunity, and relating granulocyte and monocyte functions to the patient genotype has become an important part of contemporary research. Although it is essential to move this field forward, a systematic study comparing the efficacy and suitability for functional studies of the various available protocols for the isolation of these immune cells has not been performed. Here, we compare human granulocyte functionality under three enrichment protocols: (i) Ficoll density gradient centrifugation, (ii) anti-CD15 antibody-conjugated microbeads (positive selection), and (iii) Polymorphoprep. Primary monocytes were isolated in parallel using (i) anti-CD14 magnetic microbeads, (ii) non-monocyte depletion by antibody-conjugated magnetic microbeads (negative selection), (iii) RosetteSep antibody cocktail, and (iv) the classical adherence protocol. The best results in terms of purity and cell functionality were obtained with positive selection by magnetic microbeads for both human granulocytes and monocytes. Whereas phagocytosis of Escherichia coli bacteria was identical in all isolation procedures tested, the granulocyte respiratory burst was higher in positively selected cells. In addition, different granulocyte enrichment procedures affect cell surface receptor expression to different extents. In toto, we propose that positive selection of granulocytes and monocytes be adopted as the procedure of choice for studies of human granulocyte and monocyte functions but caution investigators to be aware of possible alterations in cell phenotypes with different isolation procedures.
authors.affiliation: Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhou
authors.forename: Rajesh
authors.initials: R
authors.lastname: Somasundaram
authors.forename: Rosa F
authors.initials: RF
authors.lastname: Nederhof
authors.forename: Gerard
authors.initials: G
authors.lastname: Dijkstra
authors.forename: Klaas Nico
authors.initials: KN
authors.lastname: Faber
authors.forename: Maikel P
authors.initials: MP
authors.lastname: Peppelenbosch
authors.forename: Gwenny M
authors.initials: GM
authors.lastname: Fuhler
mesh_terms: D002469:Cell Separation / Q000379:methods*; D004926:Escherichia coli / Q000276:immunology; D006098:Granulocytes / Q000276:immunology* / Q000502:physiology; D006805:Human Experimentation; D006801:Humans; D009000:Monocytes / Q000276:immunology* / Q000502:physiology; D010587:Phagocytosis; D011971:Receptors, Immunologic / Q000096:biosynthesis; D016897:Respiratory Burst; D013048:Specimen Handling / Q000379:methods*
chemical_list: D011971:Receptors, Immunologic