Intracellular ROS Detection Protocol

For detection of intracellular reactive oxygen species (ROS), cells were seeded and treated in triplicate in 12-well plates at a density of 0.2 × 10⁶ cells per well, following which cells were serum starved for 24 hours. One ml solutions of 50 μg ml⁻¹ of us-GO, and 10 mM H₂O₂ were prepared and added to wells using a 1 in 10 dilution to give a final concentration of 5 μg ml⁻¹ us-GO, 1 mM H₂O₂ and 400 μM H₂O₂, and incubated for 4 hours. After treatment, supernatants were aspirated and cells gently washed once with 1 mL per well of prewarmed PBS (with Ca²⁺/Mg²⁺ Sigma-Aldrich, Merck Sigma, UK). Cells were detached using 0.05% Trypsin–EDTA solution (Sigma-Aldrich, UK) for 5 min, then centrifuged for 5 min at 1500 rpm; supernatants were then aspirated, and pellets containing cells were resuspended in 1 μM hydroethidine (Sigma-Aldrich, Merck Sigma, UK) for 20 min on ice. Ten thousand cells were analysed on a BD FACSVerse flow cytometer using 488 nm excitation and 620 nm band-pass filters for HE detection. Cells treated with GO, but unstained with HE, were also run in order to ensure that the detected signal was not due to the inherent fluorescence of GO.