8 ohdg assay preparation reagent set

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.

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.

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).

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.

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).