Ht 8 oxo dg elisa kit 2

The levels of 8-oxo-G in the samples were assessed using an HT 8-oxo-dG ELISA Kit II (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Fragmented DNA from each sample (500 ng) was used as the input for all ELISAs. After optical density measurements were taken at 450 nm using a microplate absorbance reader, concentrations were calculated on the basis of a linear calibration curve generated for each experiment using 8-oxo-G standard solutions. AP sites were quantified using the OxiSelect™ Oxidative DNA Damage Quantitation Kit (Cell biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, 500 ng of genomic DNA was labeled with an aldehyde-reactive probe (ARP) containing a biotin moiety, before being immobilized in a micro-well and incubated with streptavidin-conjugated HRP. The DNA was next incubated with an HRP substrate and the resulting absorbance measured at 450 nm. To infer the number of AP sites from the measured absorbance units, a standard curve was generated using serial dilutions of a stock standard solution containing 40 ARP/105-bp of DNA.

Plasma 8-OHdG levels were determined using an enzyme-linked immunosorbent assay. Peripheral blood samples (50 µL) were centrifuged (15 min at 2500× g) at room temperature. The plasma was collected and immediately analyzed according to the manufacturer’s instructions using Trevigen’s HT 8-oxo-dG ELISA Kit II (No. 4380-192-K; Gaithersburg, MD, USA). The absorbance at 450 nm of each well was determined using a Multiskan™ FC microplate reader (Thermo Scientific™, Vantaa, Finland). Product formation is inversely proportional to the amount of 8-OHdG present in the sample. The 8-OHdG levels were determined in duplicate (per sample) and according to the 8-OHdG standard curve.

Total genomic DNA was isolated according to Mullenbach et al. [53 (link)], and following previously described steps to reduce artifactual DNA damage [54 (link)]. DNA damage was quantified using two distinct methods: a PCR based assay (q-PADDA) which detects many types of DNA damage with high sensitivity [52 (link), 54 (link)], and a colorimetric based assay (HT 8-oxo-dG ELISA Kit II, Trevigen, MD) which detects exclusively 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) lesions. 8-oxo-dG is one of the major products of DNA oxidation. The primer-anchored DNA damage detection assay (q-PADDA) was performed as we previously described [52 (link)]. We chose to quantify DNA damage within the transcribed strand (TS) and non-transcribed strand (NTS) of TP53 (commonly referred as p53), because p53 is the most frequently mutated gene in human cancer [55 (link)] and is mutated in nearly all smoking related cancers.[56 (link), 57 (link)] For DNA damage quantification, as well as for all other analysis described below, we performed three independent experiments, each with 3–6 technical replicates.

HT29 cells at 70% confluency were or were not treated with a hydroxyl radical generating system (100 μM H₂O₂ and 200 μM FeSO₄ (Sigma Aldrich) in the growth medium) for the indicated period. At the end of the incubation period, DNA was isolated with QIAamp®DNA Mini Kit (Qiagen) following the manufacturer's protocol. Oxidative DNA damage was verified by determination of 8-oxo-dG levels using the HT 8-oxo-dG ELISA Kit II (Trevigen) according to the manufacturer's instructions.