期刊
CLINICAL BIOCHEMISTRY
卷 41, 期 7-8, 页码 570-575出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.clinbiochem.2007.12.021
关键词
human neutrophils; neutrophils isolation procedure; anticoagulants; respiratory burst; intracellular calcium
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. (c) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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