Abstract:
: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 microl neutrophils (3 X 10(6) cells/ml) and 50 microl of 0.5 mM lucigenin per well, in addition to either 100 microl of OZ (5 mg/ml) when zymosan was opsonized with 10-20% serum or 100 microl of PMA solution (1 X 10(-6) M) with automatic regular intervals of mixing and detection during the continuous measurement at 37 degrees 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.
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Hasegawa H,Suzuki K,Nakaji S,Sugawara Kdoi
10.1016/s0022-1759(97)00159-2subject
Has Abstractpub_date
1997-12-15 00:00:00pages
1-10issue
1eissn
0022-1759issn
1872-7905pii
S0022-1759(97)00159-2journal_volume
210pub_type
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