Analysis and assessment of the capacity of neutrophils to produce reactive oxygen species in a 96-well microplate format using lucigenin- and luminol-dependent chemiluminescence

Hiroko Hasegawa*, Katsuhiko Suzuki, Shigeyuki Nakaji, Kazuo Sugawara

*この研究の対応する著者

研究成果: Article査読

95 被引用数 (Scopus)

抄録

The chemiluminescence (CL) assay has been used to measure the reactive oxygen species (ROS)-generating capacity of phagocytes. To achieve more optimal measurement conditions for a multi-channel microplate photon-counting CL analyzer with the cooled charge-coupled device (CCD) camera which offers enhanced sensitivity, we investigated factors affecting the variability in lucigenin-dependent CL (LgCL) measurement of human neutrophils stimulated with either opsonized zymosan (OZ) or phorbol myristate acetate (PMA). We obtained sensitive LgCL responses with good reproducibility and rapid data-acquisition using 50 μl neutrophils (3 × 106 cells/ml) and 50 μl of 0.5 mM lucigenin per well, in addition to either 100 μl of OZ (5 mg/ml) when zymosan was opsonized with 10-20% serum or 100 μl of PMA solution (1 × 10-6 M) with automatic regular intervals of mixing and detection during the continuous measurement at 37°C. Furthermore, we studied the contribution of various ROS to LgCL and luminol-dependent CL (LmCL) using modulators of ROS metabolism including superoxide dismutase (SOD), catalase, deferoxamine and sodium azide (NaN3). LgCL was inhibited by SOD but not by the other agents, whereas LmCL was inhibited by NaN3 and deferoxamine. Thus, it was demonstrated that LgCL detects the superoxide anion with high selectivity whereas the LmCL assay measures myeloperoxidase (MPO)-mediated formation of hypochlorous acid. Such microplate-based multiple measurements facilitate the accurate assessment of phagocytic function.

本文言語English
ページ(範囲)1-10
ページ数10
ジャーナルJournal of Immunological Methods
210
1
DOI
出版ステータスPublished - 1997 12 15
外部発表はい

ASJC Scopus subject areas

  • 免疫アレルギー学
  • 免疫学

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