Optimization of nb-4 and hl-60 differentiation for use in opsonophagocytosis assays.
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.
Fleck
Roland A
RA
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
Athwal
Harpinder
H
Bygraves
Jane A
JA
Hockley
David J
DJ
Feavers
Ian M
IM
Stacey
Glyn N
GN
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*
D039481:CD11b Antigen; D009895:Opsonin Proteins; D014212:Tretinoin; D004126:Dimethylformamide