Luminol

Suspension-cultured cells (100 mg) in modified liquid N6 medium and two leaf strips in sterile water were treated with the following elicitors at the indicated final concentrations: 500-fold diluted OS_MYL, 100-fold diluted dialysate of OS_MYL, 100 nM OsPep3, 100 nM OsPep4, or sterile Milli-Q water (as mock treatment). OsPep3 and OsPep4 were purchased from Pepmic (Suzhou, China) following custom synthesis. H₂O₂ concentration was measured at the indicated times using a previously described luminol-dependent chemiluminescence assay using luminol (Nacalai Tesque, Kyoto, Japan) and a TD-20/20 luminometer (Turner Designs, San Jose, CA, USA) [35 (link),69 (link),71 (link)]. A standard curve was constructed to calculate the concentration of H₂O₂. To assess the inhibitory activity of OS_MYL against chemiluminescence, 500-fold diluted OS_MYL and/or 2 µM H₂O₂ were mixed with sterile water. The mixtures were incubated at room temperature for 5 min before being subjected to the luminol-dependent chemiluminescence assay.

Bone marrow cells from WT and Mincle^−/− mice were cultured in 10% FCS/RPMI supplemented with 50 ng/mL GM-CSF (BioLegend) for eight days. Attached cells were detached by 1 mM EDTA-supplemented PBS and were used as BMDMs. The cells were added into 96-well plates coated with 8 nmol/well of AF-2 or TDB, or 100 μg/mL zymosan (SIGMA), in the presence of 300 μM luminol (Nacalai tesque) for 60 min. Luminescence was quantified by POWERSCAN HT (DS Pharma Biomedical). Data are presented as mean ± SE of triplicate assays.

Luminol, dithiothreitol, bovine serum albumin (BSA), NaCl, NaHCO₃, Na₂CO₃, H₂SO₄, HCl, CH₃COONa, Na₂HPO₄, and CH₃COOH were acquired from Nacalai Tesque (Kyoto, Japan). KCL, tween 20, hydrogen peroxide, p-iodonitrotetrazolium (INT), sodium borohydride, and KH₂PO₄ were sourced from Wako Pure Chemical Industries (Osaka, Japan). Anti-rabbit IgG-biotin conjugate and avidin were obtained from the following USA companies: ImmunoReagent Inc. (Burlington, NC, USA) and Calzyme Laboratories Inc. (San Luis, CA, USA), respectively. Biotin-hydrazide and NaIO₄ were purchased from Sigma-Aldrich (St. Louis, MO, USA), while sodium hydroxide was from Merck (Darmstadt, Germany). Dextran 40 was obtained from TCI (Tokyo, Japan). Doxorubicin HCl was purchased from Funakoshi co (Tokyo, Japan). Double distilled water was used throughout the whole experiment, and it was produced from Yamato Autostill WG203 (Tokyo, Japan).
By combining sodium chloride, potassium dihydrogen phosphate, disodium phosphate, and potassium chloride, phosphate buffer saline (PBS, 100 mM pH 7.4) was prepared. A 50.0 mM carbonate-bicarbonate buffer with pH 9.6 was made from Na₂CO₃ and NaHCO₃.