Graphene Oxide and H2O2 Induced Cell Damage

On the 10th day of proliferation, cells were trypsinized. The next step was counting the cells using a Scepter Cell Counter (Merck Millipore, Darmstadt, Germany) and transferring 30,000 cells/well to Cellware 6-well plate covered by Collagen I (Greiner Bio-One, Monroe, NC, USA). When the cells obtained 80% confluence, the proliferation medium was replaced by differentiation medium (2% HS/ Dulbecco’s Modified Eagle Medium (DMEM)/AB). After the 2nd day of differentiation, 0.125 µM of GO (TCI Chemicals, Portland, USA), dissolved in 0.04 µL/mL DMSO as a vehicle, which was also used in the control medium, was added for 24 h. The MTT assay was used to choose the concentration of GO and H₂O₂ [25 (link)]. During the last part of the experiment (the last one hour of GO incubation), H₂O₂ (3 mM, Sigma Aldrich, Poznań, Poland) was added. The main purpose of H₂O₂ administration was to cause cell damage (Figure 1).