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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fc gamma and complement receptors

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Author(s):
Santos, Everton O. L. [1, 2] ; Azzolini, Ana Elisa C. S. [2] ; Lucisano-Valim, Yara M. [2]
Total Authors: 3
Affiliation:
[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Bioquim & Imunol, BR-14040903 Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Quim & Fis, BR-14040903 Ribeirao Preto, SP - Brazil
Total Affiliations: 2
Document type: Journal article
Source: JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS; v. 72, p. 67-71, MAR-APR 2015.
Web of Science Citations: 6
Abstract

Introduction: 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 Fc gamma and complement receptors (Fc gamma R 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 Fc gamma R, CR, and Fc gamma R + 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 x 10(6) cells mL(-1) and 40 mu g mL(-1) FITC-labeled ICs, incubated for 30 min, at 37 degrees C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH 4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. Discussion: 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 Fc gamma R 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. (C) 2014 Elsevier Inc. All rights reserved. (AU)

FAPESP's process: 07/06925-5 - The mechanisms of action of flavonoids on the metabolism oxidative and on the phagocytosis from human neutrophils triggered by Fc-gamma and complement receptors
Grantee:Everton de Oliveira Lima dos Santos
Support Opportunities: Scholarships in Brazil - Master
FAPESP's process: 07/02487-3 - Study of the mechanism of action of coumarin derivatives on the myeloperoxidase activity: investigation about metabolization of these compounds to phenoxyl free radicals, implications on the oxidative stress and modulation of leukocyte functions
Grantee:Yara Maria Lucisano Valim
Support Opportunities: Regular Research Grants