Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

• Type: Article in International Scientific Journal
• Date: 2008

Authors

Marisa Freltas (Author) Other; Graca Porto (Author) ICBAS; Jose L F C Lima (Author) FFUP; Eduarda Femandes (Author) FFUP

Journal

Title: Clinical Biochemistry

Vol. 41

Pages: 570-575

ISSN: 0009-9120

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 42 Citations

Scopus - 79 Citations

Scientific classification

FOS: Engineering and technology > Medical engineering

Other information

Authenticus ID: P-003-ZJW

DOI: 10.1016/j.clinbiochem.2007.12.021

Abstract (EN): Objectives: To assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA). Design and methods: Isolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca2+](i)) was measured using Fluo-3 AM, a calcium-sensitive dye. Results: EDTA provided the highest number of isolated neutrophils/mL of blood (1.7 x 10(6)+/- 1.5 x 10(5)) when compared with citrate (0.46 x 10(6)+/- 0.95 x 10(5)) and heparin (0.66 x 10(6)+/- 0.15 x 10(5)). EDTA originated less degree of PMA-induced activation (370 +/- 30%) relatively to citrate (830 +/- 98%) and heparin (827 +/- 77%). [Ca2+](i) was lower with EDTA (122 +/- 11 nM) when compared with citrate and heparin (150 +/- 13 and 230 +/- 30 nM). Conclusion: The anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 6