How OxyFLOW™ Detects Oxidative DNA Damage
The cell suspension is fixed and permeabilized to allow the binding protein to enter the cell. If lympho-hematopoietic cells are used, the fixative supplied with the kit also contains a lysis buffer. The cells are incubated with the fluorochrome-conjugated binding protein for 45 minutes. After washing, the cells can be incubated with other fluorochrome-conjugated proteins or the fluorescence intensity of the FITC-labeled binding protein detected directly. OxyFLOW™ is standardized and validated prior to evaluating samples using a methylene blue dose response. In the presence of bright light illumination, methylene blue causes single strand breaks in DNA due to oxidative DNA damage. Increased incorporation of the FITC-conjugated binding protein results in an increase in fluorescence intensity which can best be graphically represented using flow cytometric overlay histograms as shown in the diagram below.
The cytotoxicity produced by compounds that cause oxidative DNA damage, e.g. etoposide, can be correlated using either HALO®, LUMENESC™ or LumiSTEM™ depending on the type of target cells being employed. For example, the graphs below show the effect of etoposide. This drug causes double-stranded DNA breakages. Human peripheral blood mononuclear cells were incubated with increasing doses of etoposide in suspension culture for 4 and 7 days. Thereafter, the cells were removed. The effect of etoposide on DNA at 4 days is shown in the overlay flow cytometric histogram. The corresponding cytotoxicity is shown in the the upper left HALO®-96 MeC dose response curve. Although the solvent used, DMSO, results in oxidative DNA damage, the shift to the right in the median fluorescence intensity with increasing doses of etoposide indicates that the cytotoxicity seen in the HALO® assay is due to oxidative DNA damage. At higher doses of etoposide, the median fluorescence intensity decreases, indicating cell death, which is also observed at these doses in the HALO® assay. Despite a dose response being detected by HALO® on day 7 of incubation, the response is extremely low, indicating that few cells have survived.
