DMPO  别名：5,5-二甲基-1-吡咯啉-N-氧化物

DMPO is a water soluble nitric oxide spin trap; allows the measurement of oxygen-centered free radicals in biological systems at room temperature using electron spin resonance (ESR). Has a high reaction rate constant for superoxide and hydroxyl radicals, and distinguishes simultaneously among a variety of important biologically generated free radicals.

Quantitation of Spin Probe-Detectable Oxidants in Cells Using Electron Paramagnetic Resonance Spectroscopy

Materials
Diethylenediamine pentaacetic acid (DTPA), deferoxamine mesylate (DFO), diethyldithiocarbamate (DETC), phorbol-12-mysirate13-acetate (PMA), dimethyl sulphoxide (DMSO), 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride (CMH), 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP•), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), Dublecco's Modification of Eagles Medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS), and 10,000 units/mL penicillin and 10,000 μg/mL streptomycin.

Cell Culture
RAW264.7 murine macrophage cell line was cultured in DMEM supplemented with 10% FBS as well as 100 units/mL penicillin and 100 μg/mL streptomycin.

Sample Preparation
Buffer was added to the weighed solid to directly achieve the concentration of 0.2 or 0.5mM for CMH and of 50mM for DMPO. For the metal chelator effects, 0.5M CMH stock solution was made in PBS first, and then immediately diluted in PBS containing respective metal chelators to a final concentration of 0.5mM. A timer was started when the buffer was added to solid. Cells were washed twice with PBS and collected via scrapping in PBS containing 1mM DTPA, pH 7.4 (PBSD). Following centrifugation at 300 × g for 5 minutes, the cell pellet was resuspended in PBSD containing CMH or DMPO. For the experiments involving stimulation, PMA or the same volume of DMSO (<0.5‰ of volume) was added to cell resuspension. Cell concentration and viability were determined before and after the experiment by trypan blue exclusion and TC20 automated Cell Counter.
Samples were kept at room temperature (RT) (or, on ice or at 37°C for temperature effect), and open to the air (or sparged with nitrogen for at least 20 min for oxygen effect). Samples containing cells were mixed from time to time by gently inverting the container upside down. Aliquots were either injected into Aqua-X sample cell for EPR measurement at RT, or transferred into EPR quartz tubes, frozen in liquid nitrogen, and stored at −80°C until analyzed at 150K. For fresh samples, the time was recorded when the EPR run started; for frozen samples, when the EPR tube was merged into liquid nitrogen.

EPR Measurements
EPR spectra were recorded by Elexsys E500 equipped with 4122SHQE-W1 resonator. The instrumental settings were the same as those have been extensively applied in previous studies using spin probes. For RT measurements they were: 2 G modulation amplitude, 40.96 ms time constant, 81.92 ms conversion time, 20 mW power and 1 scan. For measurements at 150K, the temperature was maintained by variable temperature control unit, and the instrumental settings were: 5 G modulation amplitude, 40.96 ms time constant, 81.92 ms conversion time, 2.0 mW power, and 4 scans were accumulated.

Data analysis
Spectra were analyzed using the XEPR software bundled with the instrument. Since the EPR spectrum is the first derivative of the actual absorption spectrum, spectra were first integrated to obtain the absorption spectra. This was followed by baseline correction and subsequent second integration that yielded the area under curve (AUC) in arbitrary units (AU). The AUC was then used to calculate the concentration of CM• or DMPO-OH• based on the CP• standard curve. As shown in the Results, 150K spectra had much lower intensity than RT ones. The baseline of the actual absorption spectra acquired by the first integration was very irregular and the intensity was so low compared to the baseline noise that the AUC generated by the second integration varied largely. Instead of AUC, the difference between the peak and the trough in the EPR spectra (Peak to Trough) has been extensively used for quantitation of signal intensity. We found that the method of Peak to Trough provided much lesser variability (based on R²) for CP• standard curves compared to the quantitation using AUC. Therefore, the quantitation for 150K measurements was attained using the method of Peak to Trough (in AU). Another common method is to simulate the spectra then integrate.