Fac strak

An annexin V/propidium iodide (PI) assay (Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis Kit with AlexaFluor 488 Annexin V and PI for flow cytometry, Life Technologies) was performed to examine whether cell death had occurred by apoptotic or necrotic pathways. After a 24 h incubation of U87 glioma cells in 75 mL flasks (1×10⁶ cells per flask), the medium was removed, and HNC samples were added at 50 µg/mL. After a further 24 h incubation, the medium was removed, and the cells were washed in ice-cold PBS. Harvested cells were suspended in 100 µL annexin-binding buffer (Invitrogen, Carlsbad, CA, USA), and subsequently, 5 µL of annexin V linked with Alexa Fluor 488 and 1 µL of PI were added (Invitrogen). Cells were analyzed using FAC-Strak (Becton-Dickinson, Germany; software – SimulSet), measuring the fluorescence emission at 530 and 575 nm using excitation at 488 nm.

An Annexin V/PI assay (Alexa Fluor^® 488 Annexin V/Dead Cell Apoptosis Kit with Alexa Fluor 488 Annexin V and PI for flow cytometry, Life Technologies) was performed to examine whether the cell death occurred by apoptotic or necrotic pathways. After 24 hours of incubation of U87 and U118 glioma cells in 75 mL flasks (1×10⁶ cells per flask), the medium was removed, and GO and rGO samples were added at 100 μg/mL. After further 24 hours of incubation, the medium was removed and the cells were washed in ice-cold phosphate-buffered saline. Harvested cells were suspended in 100 μL of Annexin binding buffer (Invitrogen, Carlsbad, CA, USA), and subsequently 5 μL of Annexin V linked with Alexa Fluor 488 and 1 μL of PI were added (Invitrogen, Carlsbad, CA, USA). Cells were analyzed using FACStrak (Becton Dickinson, Heidelberg, Germany; SimulSet software), measuring the fluorescence emission at 530 nm and 575 nm (or equivalent) using excitation at 488 nm.