Influence of ROS on CO effect at mitochondrial level. A. Mitochondria were pre-treated or not with β-carotene, followed by CO at 0, 10, 50, 100 or 250 μM or H2O2 at 500 μM treatment. ROS quantification using 2',7'-dichlorofluorescein diacetate (H2DCFDA, λexc: 485 nm, λem: 530 nm) was done in isolated mitochondria. The values are expressed in relative percentage of 500 μM of H2O2 (100%) at 28 minutes after treatment. All values are mean ± SD, n = 3; * p < 0.05 compared to control mitochondria and ** p < 0.05 compared to mitochondria treated with CO at 50 μM. Mitochondria were pre-treated in the presence or absence of 1 μM of β-carotene and/or 10 μM of CO, then atractyloside at 300 μM or calcium at 5 μM was added, followed by swelling measurements (absorbance at 540 nm) for B and depolarization measurements (Rhodamine 123, λexc: 485 nm, λem: 535 nm) for C. In B and C the values are expressed in relative percentage of 5 μM of Ca2+ (100%) measured at 15 minutes after treatment. All values are mean ± SD, n = 3; * p < 0.05 compared to control mitochondria, ** p < 0.05 compared to Ca2+-treated mitochondria, *** p < 0.05 compared to Atra-treated mitochondria, # p < 0.05 compared to β-c and Ca2+-treated mitochondria and ## p < 0.05 compared to β-c and Atra-treated mitochondria. D. Inner membrane permeabilization was assessed according to . Mitochondria were pre-treated 1 μM of β-carotene and 10 μM of CO, followed by addition of calcium at 5 μM. Data was acquired at 412 nm for 20 minutes at 37°C.