Superoxide dismutase sod

Quercetin, protoporphyrin IX (PpIX), dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, USA. Ethanol, superoxide dismutase (SOD), catalase, ethidium bromide, hypoxanthine, diethylenetriaminepentaacetic Acid (DTPA), trifluoroacetic acid, acetonitrile, iron(II) perchlorate, hydrogen peroxide, fluorescein, phosphoric acid, Dulbecco’s phosphate-buffered saline (D-PBS) were purchased from FUJIFILM Wako Pure Chemical Corporation., Japan. 3’-(p-aminophenyl) fluorescein (APF) was purchased from Sekisui Medical Co. Ltd., Japan. Dihydroethidium DHE was purchased from Invitrogen, CA. Compound libraries (Core library; for pilot screening) were provided by drug discovery initiative (DDI), the University of Tokyo (https://www.ddi.u-tokyo.ac.jp/en/). WST-8 was purchased from Dojin Laboratoies, Japan. Xanthine oxidase was purchased from Calbiochem (USA).

Dimethyl sulfoxide (DMSO), N-formyl-methionyl-leucyl-phenylalanine (fMLP), 4-aminobenzoic acid hydrazide (ABAH), phorbol 12-myristate 13-acetate (PMA), hypoxanthine and xanthine oxidase were purchased from Sigma-Aldrich Japan (Tokyo, Japan). 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one hydrochloride (MCLA) was purchased from Tokyo Kasei (Tokyo, Japan). Superoxide dismutase (SOD) and sodium hypochlorite solution were purchased from Wako Pure Chemical Industries (Osaka, Japan). Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) were purchased from GORYO Chemical (Sapporo, Japan).
fMLP (1 mM) and PMA (0.1 mM) were prepared by dissolving into DMSO and stored at -80°C as stock solutions. They were diluted with Ringer-Hepes buffer (RH buffer: 154 mM NaCl, 5.6 mM KCl, and 10 mM Hepes, pH7.4) just prior to use: fMLP [1:3 (v/v)] and PMA [1:20 (v/v)]. ABAH, SOD and hypoxanthine were dissolved in RH buffer. Stock MCLA was dissolved in Milli-Q water and its concentration was adjusted according to the molar extinction coefficient after filtration.

To cancel out the inactivation effect of reactive species, the inactivation effects of individual radical scavengers, which react with various reactive species, were investigated. If the inactivation effect is reduced by adding a radical scavenger, the factor of the reactive species can be deduced. In this study, 10 mM of dimethyl sulfoxide (DMSO; Nacalai Tesque, Inc., Kyoto, Japan) as an OH radical scavenger [16 ], 10 mM of sodium azide (Wako Pure Chemical Industries, Ltd.) as a singlet oxygen scavenger [17 ], 300 U/ml of superoxide dismutase (SOD; Wako Pure Chemical Industries, Ltd.) as a superoxide scavenger [18 (link)], and 300 U/ml of catalase (Sigma-Aldrich Japan K.K., Tokyo, Japan) as an H₂O₂ scavenger [19 ] were prepared in 200 μl of PBS containing >10⁶ of P. aeruginosa. The suspensions were treated with carbon dioxide plasma and nitrogen plasma and the surviving bacteria were measured by counting of CFUs (n = 3).