Genomic DNA was extracted after plasma exposure and media in a 48 well plate (1 ml per well) was exposed to D2O + N2 plasma for 0 (control), 3 min and 0.5 µM of actinomycin D for OH-dG detection, 1 % agrose gel and CD analysis. Treated cells were subjected to genomic DNA extraction kit. Genomic DNA was extracted following a standard molecular biology protocol and re-suspended in 50 µl water24 (link). The same amount of genomic DNA (2 mg) extracted from cells was used for the detection of 8-OH-dG level using a oxidative DNA damage ELISA kit (Cell Biolabs, inc. USA), same amount of DNA was loaded on a 1 % agarose gel and runned for 1 h. Then, the DNA bands were photographed, after staining with ethidium bromide.
Oxidative dna damage elisa kit
Manufactured by Cell Biolabs
Sourced in United States
The Oxidative DNA Damage ELISA Kit is a quantitative, in vitro assay designed to measure the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker for oxidative DNA damage. The kit utilizes a competitive ELISA format to determine the concentration of 8-OHdG in samples.
Automatically generated - may contain errors
7 protocols using oxidative dna damage elisa kit
1
Oxidative DNA Damage Detection
2
Oxidative DNA Damage Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Determination of 8-OHdG concentrations was performed using the Oxidative DNA Damage ELISA Kit from Cell Biolabs (San Diego, CA) according to their instructions.
3
Quantification of Cellular Redox Status
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 24 h post treatments, we washed the spheroids twice with PBS and incubated them in 50 µL TrypLE express reagent (12604-021, Life Technologies Europe B.V., Bleiswijk, The Netherlands) for 15 min at 37 °C in a 96-well plate. Immediately after, we added fresh warm culture medium and we dissociated the spheroids with gentle mechanical force using a Pasteur pipette. After the cells were centrifuged at 1000 rpm up to 3 min, we measured the glutathione (GSH) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels. We quantified the total GSH and genomic DNA in 104 cells from dissociated spheroids. We measured the GSH level using a luminescence-based assay (Promega, Madison, WI, USA) following a standard molecular biology protocol [37 (link),38 (link)]. We used the genomic DNA extracted from the same number of tumor cells for the detection of the 8-OHdG level using an oxidative DNA damage ELISA kit (Cell Biolabs, inc. San Diego, CA, USA).
4
Assessing Antioxidant Status: Enzymatic and DNA Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To assess antioxidant status, the activities of glutathione reductase and SOD were measured in cell lysates or dialysate effluent using Glutathione Assay Kit (Cell Biolabs, San Diego, CA, USA) and Superoxide Dismutase Activity Assay (Cell Biolabs), respectively. Glutathione content was expresses as reduced/oxidized glutathione (GSH/GSSG). 8-hydroxy-2′-deoxyguanosine (8-OH-dG) content in dialysate were measured using Oxidative DNA Damage ELISA Kit (Cell Biolabs).
5
Quantifying Oxidative DNA Damage via ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The oxidative DNA damage ELISA kit (Cell Biolabs) is a competitive enzyme immunoassay available for rapid detection and quantification of 8-hydroxydeoxyguanosine (8-OHdG), a ubiquitous marker of oxidative stress and a by-product of oxidative DNA damage from cellular DNA samples53 (link). The quantity of 8-OHdG in an unknown sample is determined by comparing its absorption with that of a known standard curve. DNA was isolated from the treated and untreated samples using a bacterial DNA isolation kit (Promega) and equal amounts of DNA samples were analyzed for the detection of the 8‐OHdG level using the oxidative DNA damage ELISA kit.
6
Quantification of Oxidative DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
8-Hydroxy-2′-deoxyguanosine (8-OhdG) was measured with Oxidative DNA Damage ELISA Kit (Cell Biolabs, San Diego, Calif.) as per the manufacturer’s instructions. Briefly, 96-well plates were coated with 8-OHdG-BSA/PBS conjugate overnight at 4°C. After washes, the plates were blocked with assay diluent for 1 hour at room temperature. Samples and standards were incubated for 10 minutes, followed by incubation with anti-8-OHdG antibody for 1 hour. After washing, horseradish peroxidase–coupled secondary antibody was incubated for 1 hour. After several washes, peroxidase-substrate solution was added. The reaction was stopped after 5 minutes, measurements were taken at 450 nm on the Synergy-H1 plate reader (Biotek, Winooski, Vt.), and the readings were standardized to total DNA.
7
Quantifying Oxidative DNA Damage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment of cells with 3,4-DHBA in the presence or absence of 0.5 mM H2O2, DNA was isolated from cell culture using a nucleic acid purification kit (Toyobo Co., Osaka, Japan) according to the manufacturer’s protocol. Isolated DNA was then digested with nucleases to obtain 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the nucleoside form. 8-OHdG formation was determined using an oxidative DNA damage ELISA kit (Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s protocol. The absorbance of each well was recorded at 450 nm using the microplate spectrometer; cells not treated with H2O2 were used as the control. The 8-OHdG level was expressed as a percentage relative to the level in the control. The data are expressed as the mean±SD of triplicate samples. *P<0.05 compared with the H2O2-treated control cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!