Nucleostain dna damage quantification kit

DNA was extracted from the cells detached by the proposed method or trypsinization after 10 passages. A DNA extraction kit (Get pureDNA Kit-Cell, Tissue, Dojindo, Tokyo, Japan) was used to extract the DNA. In the extracted DNA, some of the cells detached by trypsinization was irradiated with UV (Mineralight XX-15S, Analytic Jena, Jena, Germany) for 30 min as a positive control. In order to evaluate the DNA damage, the number of apurinic/apyrimidinic (AP) sites in the DNA in the cells detached by either the proposed method, trypsinization, or UV irradiation was measured by using an AP site counting kit (-Nucleostain- DNA Damage Quantification Kit, Dojindo, Tokyo, Japan).

Mitochondrial DNA and nuclear DNA were isolated by using a mitochondrial DNA isolation kit (Biovision) and a DNA Extraction WB kit (Wako Pure Chemical Industries, Osaka Japan), respectively. The isolated DNA was labeled with N-Amino-oxymethylcarbonyl-hydrazino-D-biotin by the manufacturer's instructions (Nucleostain DNA damage Quantification Kit; Dojindo). OD values at 650 nm were measured with a microplate reader (Sunrise). For 8-OHdG Assay, the isolated DNA was digested with nuclease P1 followed by the manufacturer's instructions (8-OHdG Assay Preparation reagent set; Wako Pure Chemical Industries). The amounts of 8-OHdG were quantified using high performance liquid chromatography-electrochemical detector (HPLC-ECD) as described previously [33 (link)].