title: Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays.
abstract: Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D(3) and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.
authors.affiliation: Division of Cell Biology and Imaging, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, United Kingdom EN6 3QG. rfleck@nibsc.ac.uk
authors.forename: Roland A
authors.initials: RA
authors.lastname: Fleck
authors.forename: Harpinder
authors.initials: H
authors.lastname: Athwal
authors.forename: Jane A
authors.initials: JA
authors.lastname: Bygraves
authors.forename: David J
authors.initials: DJ
authors.lastname: Hockley
authors.forename: Ian M
authors.initials: IM
authors.lastname: Feavers
authors.forename: Glyn N
authors.initials: GN
authors.lastname: Stacey
mesh_terms: D000704:Analysis of Variance; D039481:CD11b Antigen / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects*; D004126:Dimethylformamide / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells / Q000648:ultrastructure; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009895:Opsonin Proteins / Q000276:immunology*; D010587:Phagocytosis / Q000276:immunology*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D039481:CD11b Antigen; D009895:Opsonin Proteins; D014212:Tretinoin; D004126:Dimethylformamide