Plication of PMA into the buffer fluid, resulting in a concentration of 10 . For these investigations either C57/BL6 mice (= wildtype control) or mice lacking the NADPH oxidase subunit gp91phox (= p91phox-/-). Mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). ESR measurements Oxidation of the spin probe CPH by superoxide forms the nitroxide CP radical. The triple-line spectrum of CP radical was detected by ESR spectroscopy, using a MS 100 spectrometer (Magnettech, Berlin, Germany). The ESR measurements were performed in field scan with the following settings: microwave frequency 9.78 GHz, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 18 mW. All samples, both from the in vitro experiments or from the venous outflow of the isolated lung, were made in 50 glass capillary tubes and measured immediately at room temperature. The ESR amplitude is in proportion to the amount of CP? reflecting the interaction of ROS with CPH [29,34,35]. Thus, the quantity of trapped ROS was directly calculated from the ESR spectrum of the probe, while the contribution of superoxide radical to the formation of CP was determined in parallel experiments performed in the presence of SOD in the buffer fluid (150 U/ml). The first sample was taken 5 min after CPH addition to the buffer fluid (time set at zero), followed by further sampling every 5 or 30 minutes, as appropriate. The values after PMA addition were assessed every minute in the respective experiments. In the isolated mouse lung samples were taken every 2 min. For quantification, the second-field component of the ESR spectrum was used. To standardize values, the amplitude of this component was divided through the receiver gain. Statistical analysis Data are given as mean ?standard error (SEM). For comparison of two groups, a two-tailed t-test was employed. For multiple comparisons, analysis of variance was used, followed by the Student-Newman-Keuls post hoc test when differences were indicated. Statistical significance was assumed when p < 0.05.Page 3 of(page number not for citation purposes)Respiratory Research 2005, 6:http://respiratory-research.com/content/6/1/ResultsWhen CPH (1 mM) was dissolved in Krebs-Henseleit buffer ESR spectroscopy resulted in a triple band PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27872238 spectrum (Fig. 1A). The ESR signal increased to 87.06 ?11.17 arbitrary units, (AU, n = 8) within 5 h when incubated in-vitro at room temperature (Fig. 1B). The presence of the iron chelating agent DFO at a concentration of 20 or 2 mM, from the beginning of the experiment, reduced the ESR amplitude 8-fold to 11.66 ?0.44 (n = 5) or 9.28 ?0.57 (n = 4) AU within 5 h, with no major difference being observed between the high and the low concentration of DFO (Fig. 1B). Pre-incubation of the buffer with the copper chelator DETC (5 ) further reduced the increase in signal (data not shown). In line with these in vitro data, ESR signal intensity increased when isolated rabbit lungs were perfused under normoxic LurbinectedinMedChemExpress PM01183 ventilation with CPH (1 mM) for 2.5 h (Fig. 2). Perfusion with FeCl2 (1 ) resulted in a markedly higher ESR signal intensity (Fig. 2). Addition of H2O2 to the buffer fluid (8 ol/ min, started after 1.5 h), in the presence of FeCl2, induced a strong increase in ESR signal intensity, reaching a value of 707.14 ?103.15 AU (n = 4) within 1 h of lung perfusion (Fig. 2). Performing a sequential mode of 30 min periods of ventilation maneuvers with different inspiratory oxygen concentrations (1 ?21.
