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The protein calprotectin (S 100A8/A9) is present at a remarkably high concentration in the cytosol of polymorphonuclear neutrophilic granulocytes (PMN), but its mechanism of action is still uncertain.Inflammatory mediators trigger PMN to produce reactive oxygen species (ROS: O2, H2O2, 7OH).Mediated by myeloperoxidase in PMN, HOCl is then formed, detectable in a luminol-dependent chemiluminescence (CL) assay;S100A8/A9 also elicits CL-but in a different way-in response to H2O2, in a cell-free system: S 100A8/A9-induced CL increased, whereas myeloperoxidasetriggered CL decreased with pH > 7.5;and myeloperoxidase needed NaCl for CL, whereas S 100A8/A9 did not.The combination of native calprotectin, recombinant S100A8 and A9, or the S100B protein with NaOCl markedly enhanced CL.However, at higher concentration of S 100A9 (plus NaOCl), but not S 100A8, the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function.In this assay system, and also with intact PMN stimulated by H2O2 or (PMA), 20 μM hydroxycobalamin (vitamin B 12) reduced the luminescence markedly.This anti-oxidant effect was present in the cell-free set-up whether myeloperoxidase, S 100A9 or S 100B was used as ROI-instigating agent.We take these findings as proof-of-principle that our cell-free luminescence system is well suited as a simple and robust assay for redox effects of drugs and biological agents.