Esp 300 epr spectrometer

This method was previously employed as an accurate measure of total radical species in different tissues^{43 (link)}. The biopsies are dissolved in a 1 mM solution of the hydroxylamine “spin trap” (bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl) decandioate dihydrochloride CAS no. 314726-62-0) and incubated at 37 °C for 5 minutes. Samples from the solution are loaded in a capillary glass tube and inserted in the cavity of a Bruker ESP 300 EPR spectrometer (Bruker Biospin S.r.l., Rheinstetten, Germany). The nitoxide spectra were recorded at the following settings: modulation amplitude = 1.0 G; conversion time = 163.84 ms; time constant = 163.84 ms; modulation frequency 100 kHz; microwave power = 6.4 mW. All specifications about instrument calibration and data elaboration were previously reported^{38 (link)}. Tests on RWPE-1 cells were carried out as the procedures described above: cells were seeded in a 6-well plate at a density of 7 × 10⁵ cells per well and collected in serum-free medium for 48 h with vitamin E or PBS. The medium was discarded and cells detached with a scraper. Cells were centrifuged at 300 × g for 5 min and suspended in 500 μL of PBS 1X + 500 μL of hydroxylamine “spin trap” (bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl) decandioate dihydrochloride (1 mM final concentration).