title: Comparing methods for ex vivo characterization of human monocyte phenotypes and in vitro responses.
abstract: Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC)(2) isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses. Furthermore, purification of monocytes using CD14(+) microbeads resulted in a loss of CD14(low)CD16(+) monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand.
authors.forename: Lisa
authors.initials: L
authors.lastname: Johnston
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand; Wellington Cardiovascular Research Group, Wellington Hospital, Private Bag 7902, Wellington, New Zealand.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Harding
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, P.O. Box 600, Wellington, New Zealand; Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, New Zealand. Electronic address: anne.laflamme@vuw.ac.nz.
authors.forename: Anne Camille
authors.initials: AC
authors.lastname: La Flamme
mesh_terms: D000328:Adult; D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers; D002469:Cell Separation / Q000379:methods; D016000:Cluster Analysis; D016207:Cytokines / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000276:immunology* / Q000378:metabolism*; D010641:Phenotype; D055815:Young Adult
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers; D016207:Cytokines; D018950:Lipopolysaccharide Receptors
keywords: Cell purification; Ex vivo activation; In vitro stimulation; Monocyte; Monocyte activation phenotype