| Full text | |
| Author(s): |
Paoliello-Paschoalato, A. B.
[1]
;
Azzolini, A. E. C. S.
[1]
;
Cruz, M. F. C.
[1]
;
Marchi, L. F.
[1]
;
Kabeya, L. M.
[1]
;
Donadi, E. A.
[2]
;
Lucisano-Valim, Y. M.
[1]
Total Authors: 7
|
| Affiliation: | [1] Univ Sao Paulo, Dept Quim & Fis, Fac Ciencias Farmaceut Ribeirao Preto, BR-14040903 Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo, Dept Clin Med, Fac Med Ribeirao Preto, BR-14049900 Ribeirao Preto, SP - Brazil
Total Affiliations: 2
|
| Document type: | Journal article |
| Source: | JOURNAL OF IMMUNOLOGICAL METHODS; v. 412, p. 70-77, OCT 2014. |
| Web of Science Citations: | 7 |
| 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 90 min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2 x 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-lCs), 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 Fc gamma RII/CD32 and Fc gamma RIII/CD16, but they did not express significant levels of Fc gamma RI/ 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. (C) 2014 Elsevier B.V. All rights reserved. (AU) | |
| FAPESP's process: | 12/23541-4 - In vitro evaluation of antioxidant and anti-inflammatory derivatives 3-phenylcoumarins in human neutrophils stimulated and in animal models of arthritis. |
| Grantee: | Micássio Fernandes de Andrade |
| Support Opportunities: | Scholarships in Brazil - Doctorate |