Epiquik 8 ohdg dna damage quantification direct kit colorimetric

The concentration of 8-OHdG was determined in extracted insect DNA using a EpiQuik™ 8-OHdG DNA damage quantification direct kit (colorimetric) (Epigentek) in accordance with the manufacturer’s instructions. Briefly, total DNA was extracted using a DNA extractor^® TIS kit (Wako Pure Chemical Industries) from termite whole bodies. DNA was bound to wells that have high DNA binding affinity. Then the 8-OHdG present in the DNA was detected by using capture and detection antibodies. An enhancer solution was used to enhance the signal followed by reading the absorbance using a spectrophotometer at 450 nm within 2–15 min. The results are expressed as relative quantification (%) to the positive control provided by the kit and normalized to the input DNA (ng). Six biological replicates were performed, each with five workers and a queen (S1 Table).

MCF-10A cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Phenol red-free DMEM/F-12 media, horse serum, penicillin/streptomycin, antibiotic/antimycotic, human insulin (Novolin R), epidermal growth factor, trypsin-EDTA (10X), Hanks Balanced Salt Solution (HBSS), and Phosphate Buffered Saline (PBS) were purchased from Invitrogen (Carlsbad, CA, USA). Cholera toxin was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). The CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay was purchased from Promega (Madison, WI, USA). The Bromodeoxyuridine Cell Proliferation (Chemiluminescent) Assay kit was purchased from Cell Signaling Technology (Danvers, MA, USA). The EpiQuik 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) was purchased from EpiGentek (Farmingdale, NY, USA). The Qiagen Genomic-tip 20/G, Genomic DNA buffer set, and proteinase k were purchased from Qiagen (Germantown, MD, USA). benzo(a)pyrene (BaP), diallyl sulfide (DAS), PeroxiDetect^TM Kit, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from Cell Signaling (GAPDH (D16H11) Rabbit mAb; #5174S) and Abcam (DNA Polymerase β; ab26343).

The biomarker 8-oxo-dG was detected in the mammary glands and liver tissues through ELISA and IHC analyses. For ELISA, 50 mg of the tissues was collected. Total DNA was extracted using a genome extraction kit (Sigma-Aldrich) and quantified by colorimetric method. Approximately 300 ng of DNA was extracted from each sample. The content of 8-oxo-dG in the total DNA was determined using the EpiQuik™ 8-OHdG DNA Damage Quantification Direct Kit (Colorimetric) (EpiGentek). The results are presented as the ratio of 8-oxo-dG-positive cells.
For IHC, mammary glands were fixed in 10% formaldehyde for at least 24 h and sliced into 4 μm sections after embedding in wax. After dewaxing, hydration, antigen retrieval, DNA hybridization, and sealing treatment, the slices were incubated overnight in anti-8-oxo-dG monoclonal antibody (1 : 250, Trevigen) at 4°C. Next, the slices were incubated in DyLight 594-labled goat anti-mouse IgG (1 : 250, Jackson) for 30 min. After washing, the slices were evaluated using a fluorescence microscope.