Hydroethidine (HE) oxidation with addition of extracellular O2.−. (A) A single bolus of O2.− was delivered to HE-loaded MPMVECs and recorded every 5 s. (B) Tracing indicating mean nuclear fluorescence of all cells in the field after addition of DMSO vehicle (control) or O2.− with or without preincubation with DIDS (200 μM). (C) Same experiment as B using HPMVECs. (D) Peak fluorescence change in MPMVECs (fold change normalized to baseline) in response to DMSO (vehicle control, n = 5), O2.− at 0.5 μM (n = 5), 2 μM (n = 5), 5 μM (n = 4), and 10 μM (n = 5), and H2O2 (500 μM; n = 3). The effects of SOD (2500 U/ml; n = 5), catalase (1000 U/ml; n = 3), and DIDS (200 μM; n = 5) were evaluated in the presence of 10 μM O2.−. (E) Mean cellular nuclear HE fluorescence after treatment with mitochondrial complex III inhibitor antimycin A (20 μM; n = 3) or the uncoupler FCCP (2 μM; n = 3).