Oxiselect oxidative dna damage elisa kit 8 ohdg quantitation

Mitochondria from treated and untreated HCT116VA and HCT116V1 cells were isolated as described above. MtDNA from isolated mitochondria was purified using a DNeasy kit (Qiagen). 8-OHdG was determined by using Oxiselect Oxidative DNA Damage ELISA Kit (8-OHdG Quantitation; CellBioLabs) according to the manufacturer’s instructions. Results were calculated according to the standard curve. The detection range was 100 pg/ml-20 ng/ml.

For evaluating oxidative DNA damage potentially due to microplastic exposure on C. rubrum, the content of 8-hydroxydeoxyguanosine (8-OHdG) was analysed. DNA was extracted from 20 mg (wet weight) of tissue randomly collected from one coral branch for each treatment (n = 3) and control (n = 3) after 10 days of experiment using DNeasy Blood & Tissue Kits (Qiagen, Valencia, CA) and following the manufacturer’s protocol. Finally, samples were kept at −20 °C before subsequent analyses. Nucleic acids extracted (2 μg) were transferred into new 2-ml tubes and incubated for 5 min at 95 °C, then rapidly chilled on ice. Samples were digested to nucleosides by incubating the denatured DNA in sodium acetate 20 mM, pH 5.2 with 2 μl of nuclease P1 (6 U/μl; Merck KGaA, Darmstadt, Germany) for 2 h at 37 °C. Each sample was then incubated with 5 μl alkaline phosphatase (1 U/μl; Roche, Mannheim, Germany) in Tris-HCl 100 mM, pH 7.5 for 1 h at 37 °C. The reaction mixtures were then centrifuged for 5 min at 6000 × g and the supernatants tested for DNA oxidation with an OxiSelect™ Oxidative DNA Damage ELISA Kit (8-OHdG Quantitation; Cell Biolabs, CA, USA). As positive control, Escherichia coli genomic DNA (2 μg) was incubated in a final concentration of 50 and 100 mM H₂O₂ overnight at 37 °C, and subsequently tested.

Levels of 8-oxo-dG, a DNA damage biomarker, were determined in total DNA isolated from cultured K1 cells using OxiSelect™ Oxidative DNA Damage ELISA Kit (8-OHdG Quantitation) (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s protocol. Total DNA was isolated from cells with DNAzol (Life Technologies). UV spectrophotometry was used for quantification of isolated DNA. The extracted DNA was then denatured and cut with nuclease P1 (Sigma-Aldrich, Saint Louis, MO, USA), followed by incubation with alkaline phosphatase. The samples were then centrifuged (6000 rds/min, 5 min) and the supernatants were used for further analysis. Fifty microliters of sample or standards (0 to 20 ng/mL of 8-oxo-dG) were applied on 96-well plate, incubated 10 min at room temperature and then 50 µL of anti-8-oxo-dG antibody was added. After an enzymatic reaction, detection was performed in Multilabel Plate Reader Victor X (Perkin Elmer, Waltham, MA, USA). Optical density at 450 nm was measured and 8-oxo-dG concentration in each sample was calculated according to standards.