**2.5. Superoxide assay in intact cells**

Human dermal fibroblasts, which predominantly express Fp I in its complex II, were obtained from Cell Applications Inc., Japan, and grown in Dulbecco's Modified Eagle Medium Nutrient

Analysis of complex II isoform expression was carried out as described previously [8]. Briefly, total RNA was isolated from cultured cells using TRIzol LS reagent (Invitrogen) according to the manufacturer's instructions. After removal of residual DNA by treating with DNAse, cDNA was synthesized from total RNA using ReverTraAce (Toyobo, Japan) and an oligo (dT) primer. DNA sequence of the Fp I subunit of the complex II was amplified using Taq DNA polymerase with the sense primer 5′tacggagcaggca 3′ and the antisense primer 5′aatggtggcgggac 3′ and that of the Fp II subunit was amplified with the sense primer 5′cggacagaggcg3′ and the antisense primer 5′aatggctggcgggat3′. The resulting PCR products were subjected to agarose gel electrophoresis, and the two isoforms were identified by the size of the PCR products in the ethidium bromidestained agarose gels scanned with the electronic transilluminator FAS III (Toyobo, Japan).

pended in mitochondria preparation buffer (250 mM sucrose, 20 mM HEPES, 3 mM EDTA, and 1 mM sodium malonate), pH 7.5, and homogenized with a power-driven glass-teflon Potter-Elvehjem homogenizer (20 passes). The homogenate was centrifuged at 700 × g for 15 min to pellet the cell debris and nuclei. The resulting supernatant was centrifuged at 14,000 × g for 15 min to sediment the mitochondria. The pellet was washed thrice with the mitochondria isolation buffer without sodium malonate, resuspended in the same buffer, and stored on ice until analysis. Integrity of the mitochondria was analyzed by citrate synthase activity in the

Hydrogen peroxide-mediated oxidation of the fluorescent probe Amplex Red (10-acetyl-

of the isolated mitochondria as described previously [31, 32]. Since Amplex Red does not enter the mitochondria, polyethylene glycol-conjugated SOD (PEG-SOD), a membrane per-

was performed in a 96-well plate maintained at 25°C. The resorufin formation rate was measured in SpectraMax Plus spectroflourometer (Molecular Devices, Sunnyvale, CA) at an

rate was calculated using a standard curve (0–1000 nmol/ml). To eliminate the fluorescence increment due to nonspecific oxidation of Amplex Red, a parallel assay was performed

presence and absence of 0.05% sucrose monolaurate (SML) in the assay mixture.

3,7-dihydroxyphenoxazine) to resorufin was used to determine the H<sup>2</sup>

meable form of SOD, was added to the reaction mixture to convert the O2

excitation wavelength of 540 nm and emission wavelength of 590 nm. The H<sup>2</sup>

O2

in the presence of catalase, an enzyme that scavenges H2

O2

flasks were harvested by trypsinization. The cells were sus-

, which is subsequently released to the cytosol. The assay

O2

produced in the presence of catalase from that in the

O2

−

generation rate

produced in the

generation

O2

, and the result was obtained

Mixture F-12 (DMEM-F12).

94 Mitochondrial Diseases

**2.3. Isolation of mitochondria**

Cells cultured in three 225 cm2

mitochondrial matrix into H2

by subtracting the amount of H2

absence of catalase.

 **assay in isolated mitochondria**

**2.4. H2 O2**

**2.2. Analysis of complex II isoform expression**

MitoSOX Red is a fluorescent probe that is targeted to the mitochondria of intact cells and oxidized by O2 − produced within mitochondria. This probe was used to detect O2 − production in the intact cells as previously described with slight modifications [33, 34]. Briefly, DLD-1 and HT-29 colon epithelial cells and the human dermal fibroblasts were cultured on cover slips and loaded with 5 μM MitoSOX Red and 50 nM MitoTracker Green (Molecular Probes, Eugene, OR) in the dark at 37°C in the CO2 incubator for 15 min. After washing the cells for three times with prewarmed phosphate-buffered saline (PBS), three cover slip cultures each from DLD-1 and HT-29 cells were treated with the vehicle (0.2% DMSO), PEG-SOD or 10 mM NPA and incubated for 30 min. Thereafter, 10 μM atpenin A5 was added to all the cover slip cultures and imaged at 488 nm/455 nm excitation and emission wavelengths for MitoTracker Green and 633/650 nm for MitoSOX Red immediately (0 min images) and after 30 min (30 min images), using a confocal laser scanning microscope (LSM 510, Carl Zeiss, Germany). The two cover slip cultures of dermal fibroblasts were treated with 0.2% DMSO for 30 min, and subsequently one was treated with 10 μM atpenin A5 and the other one was treated with 1 μM antimycin A. Image acquisition of dermal fibroblasts was done similar to those of DLD-1 and HT-29 cells.
