Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV.

Materials All cell culture supplies, as well as MitoSOX™ Red mitochondrial superoxide detection reagent, were from Invitrogen (Grand Island, NY). All other reagents were from Sigma-Aldrich (St. Louis, MO). Pyruvate was made fresh from powder (sodium salt, Sigma-Aldrich #P2256) prior to each experiment. Other reagents were diluted from pH-adjusted stocks stored at -20°C. Artificial cerebrospinal fluid (aCSF) assay medium consisting of NaCl (120 mM), KCl (3.5 mM), CaCl 2 (1.3 mM), KH 2 PO 4 (0.4 mM), MgCl 2 (1 mM), and HEPES (5 mM) was pH-adjusted to 7.4 and stored at -20°C. Glucose (15 mM) and 4 mg/ml fatty acid-free bovine serum album (Sigma-Aldrich, catalogue #A6003) were added to aCSF on the day of the experiment.

Preparation of Primary Cortical Neurons

Primary rat cortical neurons from E18 rat cortices were prepared by enzymatic trypsin dissociation [ 24 , 25 ] and cells were cultured and plated for analysis by Seahorse Bioscience XF24 microplate-based respirometry as previously described [ 26 ]. Cytosine arabinofuranoside (5 μM) added at 4 days in vitro (DIV) was used to inhibit glial proliferation. Neurons were maintained at 37°C in a humidified atmosphere of 95% air/5% CO 2 and used for experiments at DIV 10-14. All procedures were approved by the University of Maryland Institutional Animal Care and Use Committee (IACUC protocol #1109008 and #0813014) and were in agreement with the NIH Guide for the Care and Use of Laboratory Animals. O 2 consumption rate (OCR) measurements O 2 consumption measurements were made using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience) as described [ 26 , 27 ]. Briefly, cell culture medium was removed, cells were washed once with 1 ml aCSF assay medium supplemented with glucose (15 mM) and fatty acid-free bovine serum albumin (4 mg/ml), and cells were then incubated in 675 μl of aCSF in a CO 2 -free incubator. For MitoSOX pre-treatments, cells were incubated with or without 0.1% dimethyl sulfoxide vehicle or MitoSOX treatments (0.2 to 10 μM). Treatments were either removed after 10 minutes of incubation or maintained throughout the experiment as indicated. In other experiments, cells were acutely exposed to MitoSOX via injection. MitoSOX and other drugs of interest were loaded into reagent delivery chambers a, b, c, d in a 75 μl volume and injected sequentially at 10×, 11×, 12×, and 13× the final working concentration, respectively. Each assay consisted of cycles of 3 min mix, 3 min wait, and 2 min measure, and was performed at 37°C. In experiments where the exogenous electron donor N,N,N′N′-tetramethyl-p-phenylenediamine (TMPD, 0.1 mM) was used in combination with ascorbate (10 mM), the same concentrations of these chemicals were injected in a blank well on the XF24 plate at the same time the compounds were added to cells. The OCR in the blank well which contained aCSF but no cells was used to correct cellular OCR measurements for O 2 consumption due to chemical auto-oxidation following TMPD plus ascorbate treatments.

MitoSOX Imaging

Primary rat cortical neurons were plated on 2 or 4-well chamber slides (model 1 German borosilicate; Lab-Tek) at a density of either 250,000 (2-well) or 125,000 neurons (4-well). Neurons were cultured to DIV 10, and then exposed to two different imaging paradigms for MitoSOX. In the first paradigm, half of the culture medium was removed and replaced with medium containing 200 nM MitoTracker Green (for a final concentration of 100 nM), 20 μM Hoechst (for a final of 10 μM) and either 400 nM, 2 μM, 10 μM or 20 μM MitoSOX (to obtain final concentrations of 200 nM, 1 μM, 5 μM or 10 μM, respectively). Following a 10 minute incubation, neurons were then washed with dye-free aCSF three times and then immediately imaged with a Zeiss ApoTome- and AxioCamMRm Rev.3 camera-equipped AxioObserver Z1 inverted microscope (Zeiss MicroImaging), using a 100×/1.4 Plan-Apochromat objective lens. In the second paradigm, the culture medium was removed and replaced with aCSF containing 10 μM Hoechst and 100 nM MitoTracker Green. Following a 10 min incubation, neurons were then selected for live-cell imaging based on healthy tubular mitochondrial morphology and images were captured at 2 minute intervals. Exposure times were 50 ms for MitoTracker Green (using an emission wavelength of 524 nm), 250 ms for Hoechst (using an emission wavelength of 455 nm) and 25 ms for MitoSOX (using an emission wavelength of 572 nm). Following acquisition of the 6 th image (10 minutes after the initial starting baseline), FCCP was added to the medium at a final concentration of 5 μM. Following the 8 th image, MitoSOX was added to the medium to a final concentration of 10 μM and the subcellular MitoSOX distribution was imaged for 30 minutes.

Materials All cell culture supplies, as well as MitoSOX™ Red mitochondrial superoxide detection reagent, were from Invitrogen (Grand Island, NY). All other reagents were from Sigma-Aldrich (St. Louis, MO). Pyruvate was made fresh from powder (sodium salt, Sigma-Aldrich #P2256) prior to each experiment. Other reagents were diluted from pH-adjusted stocks stored at -20°C. Artificial cerebrospinal fluid (aCSF) assay medium consisting of NaCl (120 mM), KCl (3.5 mM), CaCl 2 (1.3 mM), KH 2 PO 4 (0.4 mM), MgCl 2 (1 mM), and HEPES (5 mM) was pH-adjusted to 7.4 and stored at -20°C. Glucose (15 mM) and 4 mg/ml fatty acid-free bovine serum album (Sigma-Aldrich, catalogue #A6003) were added to aCSF on the day of the experiment.