Dpi chloride

Differentiated HL-60 cells were collected and washed twice with Hank's balanced salt solution (HBSS; Invitrogen). Cells (2×10⁶/mL, 50 µL) were treated for 15 minutes at 37°C with or without 10 µM DPI chloride (Sigma-Aldrich). Cells were stimulated with 1 µM fMLP. Extracellular O₂⁻ production was measured as SOD-inhibitable chemiluminescence detected using Diogenes reagent (National Diagnostics, Atlanta, GA, USA) using a 96-well plate Luminoskan luminometer (Thermo, Waltham, MA, USA). Extracellular H₂O₂ was measured by a luminol/HRP-based chemiluminescence assay. Briefly, cells (2×10⁶/mL, 50 µL) were collected as above and treated with or without 10 µM DPI (37°C for ten minutes). An equal volume of HBSS containing 1 mM luminol and 20 U/mL HRP was added following DPI treatment. Luminescence was estimated as above. Superoxide production and H₂O₂ production was presented as integrated luminescence value (relative light unit) [70] (link).

O₂^•–-specific chemiluminescence (CL) probe, Cypridina luciferin analog (CLA; 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one) designated as CLA was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Aluminum (III) chloride hexahydrate (AlCl₃·6H₂0), zinc sulfate heptahydrate (ZnSO₄·7H₂O), gadolinium chloride hexahydrate (GdCl₃·6H₂O), and salicylic acid (SA) were from Wako Pure Chemical Industries (Osaka, Japan). Lanthanum chloride heptahydrate (LaCl₃·7H₂O) was from Kanto Chemical Co., Inc (Tokyo, Japan). DPI chloride, Evans blue, 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron), N,N^′-dimethylthiourea (DMTU), were from Sigma (St. Louis, MO, USA). Coelenterazine was a gift from Prof. M. Isobe (Nagoya University).