Synergi fusion column

For detection of superoxide, cells were washed twice with DPBS and incubated with Hank’s Balanced Salt Solution (HBSS) containing 10 μM dihydroethidium (DHE) for 60 mins. Media were collected and centrifuged at 3000 rpm for 5 mins. Fluorescence analysis of the oxidation product 2-hydroxyethidium (EOH) was performed on a Beckman-Coulter HPLC system. Samples were separated on a Synergi-Fusion column (250×4.6 mm, Phenomenex) using a gradient elution of 10% v/v acetonitrile, 0.1% trifluoracetic acid solution (TFA) as the mobile phase A and 100% acetonitrile as mobile phase B with a flow rate set at 1.00 ml/min. Runs began at 100% phase A. Phase B was increased linearly from 0% to 42% in 25 mins, then reduced to 0% during 5 mins and kept at 0% for 5 minutes. The UV detector was set at 350 nm to monitor the elution of DHE. Fluorescence detection was measured using 480 nm (excitation) and 580 nm (emission). Authentic DHE and EOH standard solutions were injected onto the HPLC to determine the retention time of the corresponding peaks. All procedures were performed in the dark or under indirect light. Quantification was performed by normalizing the integrated peak areas with protein concentrations measured using the BCA Protein Reagent (Thermo Scientific).