Effect of argon (Ar) combined with hypothermia treatment on cortical neuronal cell death after oxygen–glucose deprivation (OGD) challenge. Rat cortical neuronal cell culture was given OGD for 90 min and then exposed to Ar gas (70% Ar and 5% CO₂ balanced with O₂) or N₂ gas (70% N₂ and 5% CO₂ balanced with O₂), combined with hypothermia (33°C) for 2 h, and then in room air cell incubator at 33°C for 24 h. (A) Dual labeling of mitochondria (mito; red fluorescence) and cytochrome C (cyto C; green fluorescence) at 4 h after gas exposure; cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar: 10 μm. (B) Percentage of neurons with cyto c release at 4 h after gas exposure. (C) Cell apoptosis and necrosis are assessed by annexin V and propidium iodide (PI) staining in fluorescence-activated cell sorting. (D) Percentage of live cells (annexin V⁻ and PI⁻). Data are expressed as mean ± SD (n = 8); *P < 0.05 and ***P < 0.001. Hy = hypothermia; IC = injury control; NC = naive control.