O2k fluo led2 module

ΔΨ_mt of isolated mitochondria (0.5 or 1 mg of mouse liver or kidney mitochondria in two ml buffer medium for normoxic or anoxic experiments, respectively; 0.25 or 0.5 mg of brain mitochondria per two ml of medium (the composition of which is described in [32 (link)]); 0.25 mg of mouse heart mitochondria per two ml of medium) was estimated fluorimetrically with safranine O [51 (link)] or rhodamine 123 [52 (link)] and expressed as arbitrary units or calibrated to millivolts as described in [32 (link)], acknowledging the considerations elaborated in [53 (link),54 (link)] regarding the inhibition of respiration and unspecific binding of safranine. Fluorescence was recorded using a Hitachi F-7000 spectrofluorimeter (Hitachi High Technologies, Maidenhead, UK) at a 5-Hz acquisition rate, at 495 nm and 585 nm excitation and emission wavelengths, respectively, or the Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module, or the NextGen-O2k prototype equipped with the O2k-Fluo Smart Module, with optical sensors including an LED (465 nm; <505 nm short-pass excitation filter), a photodiode, and specific optical filters (>560 nm long-pass emission filter) [51 (link)]. The experiments were performed at 37 °C.

O2k-Fluo LED2-Module (Oroboros) together with Amplex UltraRed (10 μM) (Invitrogen, USA) and horseradish peroxidase (1 U/ml) (Thermo Fisher Scientific, USA) was used to detect mitochondrial hydrogen peroxide production. Calibration was performed by adding 60 nM steps of a standard hydrogen peroxide solution (40 μM), dissolved in dH₂O containing HCl (10 μM). H₂O₂ production was normalized to mitochondrial protein.

ΔΨm of isolated mitochondria (1 mg of mouse liver mitochondria in two ml buffer medium, the composition of which is described in^{38 (link)}) was estimated fluorimetrically with 5 µM rhodamine 123^{38 (link)}. Fluorescence was recorded using an Oroboros O2k (Oroboros Instruments, Innsbruck, Austria) equipped with the O2k-Fluo LED2-Module with optical sensors including an LED (465 nm; < 505 nm short-pass excitation filter), a photodiode, and specific optical filters (> 560 nm long-pass emission filter)^{39 (link)}. The experiments were performed at 37 °C.