N 1 naphtyl ethylenediamine dihydrochloride

Samples were spotted onto silica gel 60 F254 (Merck, Darmtadt, Germany), and the gel plates were developed twice using 2:5:1.5 nitromethane (Sigma-Aldrich, St. Louis, MO, USA):n-propyl alcohol (Samchun, Gyeonggi, Korea):water. The developed TLC plate was dipped in 0.3% (w/v) N-(1-naphtyl) ethylenediamine dihydrochloride (Sigma-Aldrich) and 5% (v/v) sulfuric acid (Duksan, Seoul, Korea) in methanol (CARLO ERBA Reagents S.A.A., Val de Reuil, France), and then baked at 121 °C for 5 min. Glucose polymers (G1–G7, of which the DPs are 1–7, respectively) purchased from Carbosynth Co. (Berkshire, UK) were used as standard sugars.

The murine monocyte/macrophage cell line P388/D1 and murine macrophage-like cell line RAW264.7 were grown in a 96-well plate in DMEM supplemented with 5% (v/v) FBS. Prior to the experiment, the medium was replaced with fresh DMEM enriched with 2% (v/v) of FBS (negative control) or supplemented with 1) 100 ng/ml lipopolysaccharide (LPS, Sigma), 2) 10 ng/ml IFN (R&D Systems), 3) 0.01–1000 nM BacSp222, or 4) BacSp222 combined with LPS or IFN . The cells were then cultured for another 24 h to induce iNOS expression and nitric oxide synthesis. The nitrite levels were measured in culture medium by a nitrate assay performed on a microplate. One hundred microliters of cultured media was incubated with 100 μl of Griess reagent (1% (w/v) sulphanilic acid/0.1% (w/v) N-(1-naphtyl) ethylenediaminedihydrochloride (Sigma) in 2.5% (v/v) H₃PO₄) at room temperature for 10 min. Then, the absorbance was measured at 545 nm using a microplate reader (PowerWave X, BioTek Instruments). Before use, the solution of BacSp222 was tested for LPS contamination using an E-TOXATE assay kit (Sigma). The measurements ascertained that the level of LPS in 1 μM bacteriocin solution was equal to or less than 3 pg/ml. As verified in a control experiment (data not shown), such a concentration did not influenced iNOS activity in the cells used.