2.6. Competitive ELISA for Determination of Extracellular 8-oxo-dG in the Media

The 8-oxo-dG concentration of media was measured using enzyme-linked immunosorbent assay, ELISA (Health Biomarkers Sweden AB, Stockholm, Sweden) [57]. Extracellular 8-oxo-dG (blood serum, cell culture medium) as well as the 8-oxo-dG content of DNA has been used as a marker for radiation-induced oxidative stress [58]. The technique to detect 8-oxo-dG using ELISA was developed by Hagdhoost et al. [58], the Genetic and Toxicology group, Stockholm University, and made it possible to analyze the nucleotide pool sanitization using various sources, including cell culture medium [58].

Briefly, media from the treated and untreated cells (neurospheres) were collected 3 hours after treatment and then freeze-dried overnight. The dried pellet was dissolved in cold distilled water, loaded on a C18 solid-phase-extraction column (Varian, Lake Forest, CA, USA) and washed with PBS (pH 7.4). Then, 8-oxo-dG was eluted with PBS, pH 7.4, containing 20% methanol, freeze-dried, and purified again. Samples were dissolved in PBS (pH 7.4), mixed with primary antibody against 8-oxo-dG (Japan Institute for the Control of Aging, Japan) and transferred to 96-well ELISA plates precoated with 8-oxo-dG. After overnight incubation at 4°C, the plates were washed with washing solution (PBS, pH 7.4, 0.02% Tween 20 and 0.1% bovine serum albumin) followed by addition of HRP-conjugated secondary antibody (goat anti-mouse IgG-HRP, Scandinavian Diagnostic Services, Sweden) and 2 hours of incubation at room temperature. Then, the wells were washed and incubated with tetramethylbenzidine liquid substrate (ICN Biomedicals Inc.) in the dark for 15 min. The reaction was terminated by adding 2M H₃PO₄ (Merck Millipore, Darmstadt, Germany). The absorbance was measured at 450 nm using an automatic ELISA plate reader. To display the levels of 8-oxo-dG as ng/million cells, the determined 8-oxo-dG level in the pooled samples (ng/ml) was multiplied with the corresponding total volume of the media collected during the experiment, divided by the total number of cells obtained.