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16 protocols using 8 ohdg assay preparation reagent set

1

Quantifying Mitochondrial and Nuclear DNA Damage

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Mitochondrial DNA and nuclear DNA were isolated by using a mitochondrial DNA isolation kit (Biovision) and a DNA Extraction WB kit (Wako Pure Chemical Industries, Osaka Japan), respectively. The isolated DNA was labeled with N-Amino-oxymethylcarbonyl-hydrazino-D-biotin by the manufacturer's instructions (Nucleostain DNA damage Quantification Kit; Dojindo). OD values at 650 nm were measured with a microplate reader (Sunrise). For 8-OHdG Assay, the isolated DNA was digested with nuclease P1 followed by the manufacturer's instructions (8-OHdG Assay Preparation reagent set; Wako Pure Chemical Industries). The amounts of 8-OHdG were quantified using high performance liquid chromatography-electrochemical detector (HPLC-ECD) as described previously [33 (link)].
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2

8-OHdG Detection Assay Protocol

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Each sample was hydrolyzed using an 8-OHdG Assay Preparation Reagent Set (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) employing the DNA obtained as described in the method of RNA/DNA/protein extraction. Subsequently, 8-OHdG was detected using High Sensitive 8-OHdG Check (Wako Pure Chemical Industries, Ltd.) in accordance with the product protocol.
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3

Quantifying DNA Damage in L929 Cells

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L929 cells were transfected with DsRed, Atxn1-86Q-DsRed or Atxn1-86Q-DsRed+HMGB1-EGFP for 48 h. DNA damage was induced in L929 cells by 8 Gy X-ray irradiation (130 KPv, 13 min 42 s, X-ray Cabinet system). Ten minutes after X-ray irradiation, cells were harvested and DNA was extracted using the DNA Extractor TIS kit (Wako, Japan). After preparation with 8-OHdG Assay Preparation Reagent Set (Wako, Japan), the amount of 8-OHdG was measured using a highly sensitive 8-OHdG ELISA kit (Japan Institute for the Control of Aging [JaICA], Japan).
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4

Quantification of Oxidative DNA Damage

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8-OHdG, a modified DNA base product generated by free radicals, is a biomarker of oxidative DNA damage [15] (link). DNA was extracted from the liver using a DNA extractor kit (DNA Extractor TIS Kit; Wako, Osaka, Japan). Hepatic 8-OHdG concentration was measured by using an enzyme-linked immunosorbent assay kit (Highly sensitive 8-OHdG Check; Japan Institute for the Control of Aging, Shizuoka, Japan) after preparation with an exclusive kit (8-OHdG Assay Preparation Reagent Set; Wako). Results were expressed as ng/mg DNA corrected against the DNA level in each sample.
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5

Quantification of Oxidative DNA Damage

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BJ cells were cultured in the 10 cm dish at a density of 5 × 105 cells/mL. After the cells adhering to the bottom of the dish, P.g. LPS (1 μg/mL) was treated or pretreated with CA-074Me (50 μM) for 1 h. After 48 h, the cells were collected and subjected to DNA extraction by DNA Extractor TIS Kit (Wako, Osaka, Japan) according to the manufacturer's protocol. The extracted DNA were calculated and prepared according to 8-OHdG Assay Preparation Reagent Set (Wako, Osaka, Japan), 200 μg/150 μL of each sample were heated at 98°C for 2 min. After chill in ice for 5 min, the samples were incubated with 19 μL acetic buffer and 10 μL of nuclease P1 solution at 37°C for 30 min. 20 μL of Tris buffer and 1 μL of Alkaline Phosphatase solution were added and incubated at 37°C for 30 min. Then, the samples were subjected to 8-OHdG ELISA kit (High sensitive-8-OHdG check; Japan Institute for the Control of Aging, Fukuroi, Japan) following the manufacturer's protocol.
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6

Highly Sensitive 8-OHdG Quantification

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Genomic DNA was isolated from extra brain using a DNA Extractor TIS Kit (Wako, Osaka, Japan). The DNA samples were diluted in TE buffer. Then, hydrolysis of the DNA was performed using the 8-OHdG Assay Preparation Reagent Set (Wako, Osaka, Japan). The hydrolysate was filtered through Amicon Ultra (Merck Millipore, Billerica, MD, U.S.A.) prior to the measurement of 8-OHdG using a Highly Sensitive 8-OHdG Check (Nikken SEIL Corporation, Shizuoka, Japan).
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7

Quantification of 8-OHdG in F. nucleatum

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After anaerobic precultivation in 6 mL of modified GAM medium at 37 °C for 20 h, the suspension of F. nucleatum cells was adjusted to OD 600 of 0.5, and aliquots of 1 mL were placed in a 12-well culture plate. The samples were then irradiated with a violet LED at 0 or 50 J/cm2. DNA extraction of the precipitates, obtained by centrifugation from the samples, was performed using the NucleoSpin Microbial DNA kit (Macherey-Nagel, Düren, Germany). The total extracted DNA from each specimen was quantified and qualified using a NanoDrop ND-1000 (NanoDrop Technologies, Thermo Scientific, Wilmington, DE, USA). Nuclease P1 was used to digest the extracted DNA, with the use of the 8-OHdG Assay Preparation Reagent Set (Wako Pure Chemical Industries, Ltd., Osaka, Japan). 8-OHdG levels were measured using a competitive ELISA kit (Highly Sensitive 8-OHdG Check, Japan Institute for the Control of Aging, Shizuoka, Japan), following the manufacturer’s instructions. The absorbance value was measured on an ELISA plate reader set at 450 nm with a SpectraMax M5 plate reader (Molecular Devices, CA, USA). Then, compared to a standard curve, the concentrations of 8-OHdG in all samples were calculated.
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8

Measuring Oxidative DNA Damage in Nrf2 Mice

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Three animals in each group were selected randomly, and kidneys of those animals were
used for the measurement of 8-OHdG. Renal DNA of
Nrf2−/−gpt delta mice and
Nrf2+/+gpt delta mice was extracted and
digested as described previously37 (link).
Briefly, nuclear DNA was extracted with a DNA Extractor WB Kit (Wako Pure Chemical
Industries). For further prevention of artifactual oxidation in the cell lysis step,
deferoxamine mesylate (Sigma-Aldrich) was added to the lysis buffer. The DNA was digested
to deoxynucleotides by treatment with nuclease P1 and alkaline phosphatase, using an
8-OHdG Assay Preparation Reagent Set (Wako Pure Chemical Industries). The levels of 8-OHdG
(8-OHdG/105 dG) were measured by high-performance liquid chromatography with
an electrochemical detection system (Coulochem II, ESA, Bedford, MA, USA) as previously
reported19 (link).
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9

Quantifying Oxidative Stress in the Cerebral Cortex

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Lipid peroxidation (LPO) in the brain was measured using a lipid hydroperoxide assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocol. In brief, cerebral cortex (ca. 50 mg) was homogenized in 500 µL of water (n=4/group). The hydroperoxide extracted from the homogenate was measured utilizing the redox reaction with ferrous ions. The resulting ferric ions were detected using thiocyanate ion as the chromogen. The absorbance of each sample at 500 nm was obtained in triplicate.
DNA in the cerebral cortex (ca. 100 mg) was extracted using an extraction kit (DNA Extractor® TIS kit, Wako Pure Chemical Industries, Ltd.) (n=4/group). The obtained DNA (ca. 20 µg) was treated with nuclease P1 and alkaline phosphatase according to the manufacturer's protocol. The level of 8-oxo-deoxyguanosine (8-oxodG) in DNA was measured in triplicate using an ELISA kit (8-OHdG assay preparation reagent set, Wako Pure Chemical Industries, Ltd).
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10

Quantifying Oxidative DNA Damage

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As a parameter of oxidative stress levels, genomic DNA was pretreated by 8OHdG Assay Preparation Reagent Set (Wako Pure Chemical, Osaka, Japan), and 8OHdG levels were measured using the Highly Sensitive ELISA kit for 8OHdG (Japan Institute for the Control of Aging, Shizuoka, Japan) according to the manufacturers’ instructions. The absorbance at 450 nm was measured using a microplate reader (Beckman Coulter, Brea, CA, USA).
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