DNA Damage ELISA Kit

1. Prepare samples and controls in 1X Assay Buffer or buffer of choice (0.5 mL at a cell density of 5 x 105 – 1 x 106 cells/mL)., 2. Reconstitute Total ROS Green with 100 µL DMSO to produce a 500X stock concentrate., 3. Dilute 1:10 by adding 900 µL 1X Assay Buffer to the 500X stock Total ROS Green to produce a 50X working solution., 4. Add Total ROS Green to each sample at 1:50. For example, add 10 µL into 490 µL cultured cells., 5. Incubate 1 hour at 37°C., 6. Treat cells with test compounds for desired period of time. Keep cells protected from light., 7. Analyze with a flow cytometer. Total ROS Green excites at 490 nm and emits at 520 nm.

8-OHdG: BSA Coated 96-Well Plate, #270, 2 Plate Covers, #271, 8-OHdG, 100 µL at 3.06 µg/mL, #6646, 8-OHdG HRP Conjugated Monoclonal Antibody, 75 µL, #6647, Sample and Standard Diluent, 50 mL, #6648, 8-OhdG Antibody Diluent, 13 mL, #6649, 10X Wash Buffer Concentrate, 50 mL, #6650, TMB Substrate, 13 mL, #6651, Stop Solution, 13 mL, #6652, Kit Manual

8-hydroxy-2-deoxyguanosine (8-OHdG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker od oxidative stress. Hydroxylation of guanosine occurs in response to both normal metabolic processes and a variety of environmental factors (i.e. anything that increases reactive oxygen and nitrogen species). Increased levels of 8-OHdG are associated with the aging process as Well a number of pathological conditions including cancer, diabetes, and hypertension. In complex samples such as plasma, cell lysates, and tissues, 8-OHdG can exist either as the free nucleoside or incorporated in DNA. Once the blood enters the kidney, free 8-OHdG is readily filtered into the urine, while larger DNA fragments remain in the bloodstream. Because of the complexity of plasma samples, urine is a more suitable matrix for the measurement of free 8-OHdG than plasma. Urinary levels of 8-OHdG range between 2.7-13 ng/mg creatine, while plasma levels of free 8-OHdG have been reported to be between 4-21 pg/mL as determined by LC/MS. ICT’s DNA Damage (8-OHdG) ELISA is a competitive assay that can be used for the quantification of 8-OHdG in urine, cell culture, plasma, and other sample matrices. The ELISA utilizes an 8-hydroxy-2-deoxyguanosine-coated plate and an HRP-conjugated 8-OHdG specific detection antibody. This produces an assay with a useful range of 0.94 - 60 ng/mL, and a sensitivity of 0.59 ng/mL. Other highlights of this kit include a quick incubation time (60 minutes), stable reagents, and a user-friendly protocol. It is important to note that the 8-OHdG antibody used in this assay recognizes both free 8-OHdG and DNA-incorporated 8-OHdG. Since complex samples such as plasma, cell lysates, and tissues are comprised of mixtures of DNA fragments and free 8-OHdG, concentrations of 8-OHdG reported by ELISA methodology will not coincide with those reported by LC-MS where the single nucleoside is typically measured. This should be kept in mind when analyzing and interpreting experimental results.