Abasic site analysis was carried out utilizing the OxiSelect Oxidative DNA Damage Quantitation Kit (AP Sites) (Cell BioLabs, San Diego, CA) according to the manufacturer’s instructions. An apurinic/apyrimidinic site (AP or abasic sites) is one of the most frequently encountered DNA lesions in cells that can be spontaneously generated or induced by exposure to oxidative stress or to genotoxic agents such as ionizing radiation (Loeb 1986 (link)). Briefly, cells were seeded and allowed to adhere for 24 hr. Cells were then treated with 0 – 300 μM UA for 24 or 48 hr. After treatment, cells were washed with 1X PBS and harvested by trypsinization. Genomic DNA was isolated with the DNeasy® total DNA isolation kit according to manufacturers’ instructions (Qiagen, Valencia, CA) and tagged with an Aldehyde Reactive Probe (ARP) to react specifically with an aldehyde group on the open ring form of AP sites. The AP sites were then tagged with biotin and detected with a streptavidin-enzyme conjugate. The colormetric intensity was immediately measured at an absorbance of 450 nm on a plate reader (Molecular Devices, Sunnyvale, CA). AP sites were then determined by comparing the absorbance with a standard curve generated by the provided DNA standard containing predetermined AP sites for n = 3 independent experiments.
Oxiselect oxidative dna damage quantitation kit ap sites
Manufactured by Cell Biolabs
Sourced in United States
The OxiSelect Oxidative DNA Damage Quantitation Kit (AP Sites) is a laboratory kit designed to quantify the level of apurinic/apyrimidinic (AP) sites in DNA samples. AP sites are a type of DNA damage that can occur due to oxidative stress. The kit provides a simple, colorimetric assay to measure the amount of AP sites present in cellular DNA.
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5 protocols using oxiselect oxidative dna damage quantitation kit ap sites
1
Quantifying Abasic Sites in Cells
2
DNA Damage Quantification Protocols
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DNA damage levels were measured by single cell gel electrophoresis (comet assay) under alkaline conditions, measuring single-strand breaks (SSBs) and/or double-strand breaks (DSBs) as previously described [28 (link)]. Abasic (apurinic/apyrimidinic) sites were evaluated using the OxiSelect™ Oxidative DNA Damage Quantitation Kit (AP Sites; Cell Biolabs, #STA-324) according to the manufacturer’s protocol.
3
Quantifying DNA Damage via AP Sites
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DNA damage induces AP sites that have missing bases (purine or pyrimidine bases). AP sites in living cells can cause cell death or severe mutations, and the number of AP sites can serve as an indicator of DNA damage caused by high-power THz radiation. To quantify AP sites in the genomic DNA of samples, we used the OxiSelect Oxidative DNA Damage Quantitation Kit, AP Sites (Cell Biolabs Inc., San Diego, CA, USA)63 (link),64 (link). The number of AP sites was determined by comparison with the standard curve fit according to the manufacturer’s protocol. The aldehyde reactive probe (ARP) in the kit was attached to an AP site, and the ARP was tagged with biotin/streptavidin-enzyme, which allows detection of the AP sites by OD values using the ELISA reader.
4
Quantifying Oxidative DNA Damage
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For assessment of oxidative DNA damage, genomic DNA was extracted from liver tissue using the AllPrep DNA/RNA Mini Kit (Qiagen, Germany). The formation of 8-hydroxy-2’-deoxyguanosine (8-oxo-dG) and apurinic/apyrimidinic (AP) sites was quantified in pooled DNA samples of five fish per tank (n = 3 tanks/group) using the commercial kits HT 8-oxo-dG ELISA kit II (Trevigen Inc., MD, USA) and OxiSelect Oxidative DNA Damage Quantitation Kit (AP sites) (both Cell Biolabs INC., CA, USA), following the manufacturer’s instructions. Results are expressed as nM 8-oxo-dG per unit DNA (ug/ul) and AP sites per 105 base pairs (bp), respectively.
5
Oxidative Stress Biomarkers in PBMCs
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The GSH/GSSG-Glo™ Assay (Promega, #V6612, Wisconsin, USA) was used to measure the reduced glutathione (GSH) to oxidized glutathione (GSSG) ratio in PBMCs, and the OxiSelect Oxidative DNA Damage Quantitation Kit (APsites) (Cell Biolabs, Inc., San Diego, CA, USA, #STA-324) was utilized to determine and quantify the levels of abasic sites in PBMCs. All experiments were performed according to the manufacturer’s protocols.
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