Dna rna oxidative damage elisa kit

Cells were seeded in 6-well plates (1 × 10⁵/well MAD11 and 2 × 10⁵/well HEC1A) and treated for 6 h with BH10 or vitamin K3 (20 μM) or equivalent volumes of vehicle (DMSO). Non-attached and attached cells were harvested, centrifuged at 300×g for 5 min, and pellets collected for DNA extraction using the GenElute Mammalian genomic DNA miniprep kit (Sigma). DNA was then concentrated by ethanol precipitation and DNA (3 μg in 20 μL) was then denatured by adding 2 μL of 0.5 M sodium acetate (pH 5.1), 0.2 μL of 1 M MgCl₂ and heating samples to 100 °C for 5 min. Samples were cooled on ice (5 min) before adding Nuclease P1 (0.375 μL of 40 ng/μL stock, Sigma N8630) and heating at 50 °C for 1 h pH was adjusted to 7.5–8.5 with 1 M Tris (0.25 μL of 1 M Tris pH 10.5). Alkaline phosphatase (Sigma) was added to each sample (0.03U per 3 μg DNA) before heating to 37 °C for 30 min followed by 100 °C for 10 min. Samples were diluted to 0.04 ng/μL using buffer from the GenElute kit and placed on ice. Samples (equiv. of 2 μg DNA in 50 μL) were analysed using the DNA/RNA Oxidative Damage ELISA kit (Cayman Chemical), according to manufacturer's instructions.

Lipid hydroperoxide, 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-nitroguanine, PARP1 activity and adenosine triphosphate (ATP) assays were performed with whole kidneys or cells using a lipid hydroperoxide assay kit (Cayman, Ann Arbor, MI, USA), a DNA/RNA oxidative damage ELISA kit (Cayman), a 8-nitroguanine DNA/RNA damage ELISA kit (Cell Biolabs, San Diego, CA, USA), a universal PARP colorimetric assay kit (Trevigen, Gaithersburg, MD, USA), and an ATP fluorometric assay kit (BioVision, Mountain View, CA, USA) according to the respective manufacturer's instructions [25 (link)].