title: Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.
abstract: BACKGROUND
  This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution.
  
  
  METHODS
  Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue.
  
  
  RESULTS
  The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 μg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils.
  
  
  CONCLUSIONS
  The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café s/n, Ribeirão Preto, SP 14040-903, Brazil; Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil.
authors.forename: Everton O L
authors.initials: EO
authors.lastname: Santos
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: Ana Elisa C S
authors.initials: AE
authors.lastname: Azzolini
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Avenida do Café s/n, Ribeirão Preto, SP 14040-903, Brazil. Electronic address: yaluva@usp.br.
authors.forename: Yara M
authors.initials: YM
authors.lastname: Lucisano-Valim
mesh_terms: D000936:Antigen-Antibody Complex / Q000502:physiology*; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D009504:Neutrophils / Q000502:physiology*; D010587:Phagocytosis / Q000502:physiology*; D017382:Reactive Oxygen Species; D011951:Receptors, Complement / Q000502:physiology*; D017452:Receptors, IgG / Q000502:physiology*
chemical_list: D000936:Antigen-Antibody Complex; D017382:Reactive Oxygen Species; D011951:Receptors, Complement; D017452:Receptors, IgG
keywords: Fluorescein isothiocyanate; Fluorescence quenching; Immune complex; Method; Neutrophil; Phagocytosis; Trypan Blue