title: Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.
abstract: In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: A B
authors.initials: AB
authors.lastname: Paoliello-Paschoalato
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: A E 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, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: M F C
authors.initials: MF
authors.lastname: Cruz
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: L F
authors.initials: LF
authors.lastname: Marchi
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil.
authors.forename: L M
authors.initials: LM
authors.lastname: Kabeya
authors.affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil.
authors.forename: E A
authors.initials: EA
authors.lastname: Donadi
authors.affiliation: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-903, Brazil. Electronic address: yaluva@usp.br.
authors.forename: Y M
authors.initials: YM
authors.lastname: Lucisano-Valim
mesh_terms: D000936:Antigen-Antibody Complex / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000175:diagnosis* / Q000473:pathology; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D005362:Ficoll / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000276:immunology; D018375:Neutrophil Activation; D009504:Neutrophils / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*
chemical_list: D000936:Antigen-Antibody Complex; D017452:Receptors, IgG; D005362:Ficoll; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D005780:Gelatin
keywords: Fc-gamma receptor; Ficoll-Hypaque; Gelatin; Neutrophils; Oxidative metabolism; Rheumatoid arthritis