HemoGenix® receives many questions regarding its OxyFLOW™ Platform. The answers to these questions might help you decide on whether to use OxyFLOW™ for your investigative needs. If you have any questions, please contact HemoGenix® at info@hemogenix.com and we will answer them as quickly as possible and perhaps add them to this list below.
Question: What is the difference between OxyDNA and OxyFLOW™?
Answer: OxyDNA is used to detect oxidative DNA damage by fluorescence microscopy. OxyFLOW™ was specifically designed by HemoGenix®, in collaboration with Biotrin International, to detect oxidative DNA damage in cell suspensions by flow cytometry. PLEASE NOTE: BD Biosciences provides an overlay histogram on their website implying that the OxyDNA kit can be used for flow cytometry. This overlay diagram is from HemoGenix® and was obtained using the protocol developed by HemoGenix®. Any flow cytometric protocol other than that provided with the OxyFLOW™ Kit is not endorsed by HemoGenix®. Other differences between OxyDNA and OxyFLO are provided in the next question.
Question: What is included with the OxyFLOW™ Kit?
Answer: The OxyFLOW™ kit contains 3 primary reagents. The first is a fixative/lysis reagent. This is primarily used for blood-containing tissues and can be omitted if the target cells are not derived from the blood-forming system. The second is a permeabilization reagent that allows the fluorochrome conjugated bonding protein to enter the cells. The third is the FITC-conjugated 8-oxoguanine binding protein that binds with the 8-oxoguanine adducts produced as a result of oxidative DNA damage. Also included is a methylene blue standard that is diluted and used according to the manual instructions. Tubes for flow cytometry and a detailed manual are also included with the kit. HemoGenix® also provides full technical service for this product.
Question: How long does the assay take to perform?
Answer: Of course this will depend on the number of samples being processed. However, in general, the minimum amount of time required to perform the methylene blue standard and a sample would be 2 to 2.5 hours, including 45 - 60 minutes to incubate the cells with the FITC-conjugated 8-oxoguanine binding protein.
Question: What flow cytometric parameter do I use to detect oxidative DNA damage by OxyFLOW™?
Answer: The best parameter to use to measure changes in oxidative DNA damaged detected using OxyFLOW™ is the median of the peak of the X-axis fluorescence intensity detected on a single histogram plot. Prepare regions around the primary event clusters on a forward and side scatter plot of the control cells and use a single histogram plot to gate on these regions. As shown in this LINK, using overlay diagrams, the displacement to the right of the median fluorescence intensity, indicates increasing oxidative DNA damage to the gated cell population.
Question: What tissues and species can be used in the OxyFLOW™ assay?
Answer: OxyFLOW™ can be used with a wide variety of different tissues from several species. Since the FITC-conjugated bonding protein is not an antibody, it can theoretically be used with virtually any tissue from a number of species, providing 8-oxoguanine aducts are produced as a result of oxidative DNA damage. OxyFLOW™ was originally developed for human blood tissues, including peripheral blood, umbilical cord blood and bone marrow. OxyFLOW™ can be used for human, non-human primate, dog, rat and mouse tissues. It can be used on numerous other primary, non-lympho-hematopoietic cells and cell lines.
Question: What anticoagulant can I used with OxyFLOW™?
Answer: Both heparin and EDTA can be used without any problem.
Question: If I'm using cells of the blood-forming system, do I need to process the cells prior to using OxyFLOW™?
Answer: It is possible to use whole tissue, but we suggest separating the mononuclear cells and using these in the OxyFLOW™ assay. (See next question).
Question: Can the presence of red blood cells interfere with the OxyFLOW™ assay.
Answer: Erythrocytes do not interfere with the assay directly if the tissue is fresh. However, if human peripheral blood, cord blood or bone marrow is left on a rocker for long periods (12-24h and above) in an anti-coagulant tube that is less than completely full, the presence of oxygen in the tube together with the rocker motion, will cause oxidative DNA damage to occur. This might produce both false positive and false negative results. We therefore suggested using fresh tissue.
Question: Can I use OxyFLOW™ on adherent cells?
Answer: Yes. Please refer to the published article by Courter, Pereira and Baird entitled "Diesel exhaust influences carcinogenix PAH-induced genotoicity and gene expression in human breast epithelial cells in culture", Mutation Research, 2007, 625:72-82.
Question: I think I know which population of cells is showing DNA damage when they are treated with our compound. How can I specifically determine which cells are being affected?
Answer: If there is an antibody that defines you cell population, then it is easy to use the multi-parameter acquisition ability of the flow cytometer to home in on these cells. When you use OxyFLOW™, you are only using 1 channel of the flow cytometer to measure the binding of the FITC-conjugated binding protein to 8-oxoguanine adducts produced as a result of oxidative DNA damage, regardless of how this damage has occurred. If you have an antibody or other expression marker that is, or can be, conjugated to a fluorochrome other than FITC, then after the primary incubation with the FITC-conjugated binding protein, incubate the cells with the second marker. After resuspending the cells, perform flow cytometry and use your gating procedures to define whether the cell population was really being damaged.
Question: How many expression markers can I use with OxyFLOW™ to specify specific cell populations.
Answer: As many as your flow cytometer allows or as many as your flow cytometer has photomultiplyers (PMTs). You can combine as many different fluorochromes as you want with OxyFLOW™, providing your flow cytometer can measure them. This is a unique capability because it allows you to study multiple populations simultaneously.
Question: Can OxyFLOW™ be combined with any other HemoGenix® Platform?
Answer: Yes. OxyFLOW™ can be combined with HALO®, LUMENESC™ and/or LumiSTEM™. The best way of doing this is to set up extra replicate cultures for one or more of the proliferation assays (HALO®, LUMENESC™, LumiSTEM™). If the target cells are derived directly from animal studies, prepare the cells and instead of setting up the normal complement of replicates (6 or 8), set up double as many replicates. If cells are treated directly in culture, again setup double as many replicates as you would normally do. After incubation, half of the replicates will be used to measure the proliferation/cytotoxicity status of the cells. The other half will be used to remove the cells from the wells and pool them. Determine a nucleated cell count and prepare tubes for OxyFLOW™ using at least 250,000 cells/tube. If you want to incorporate multi-fluorochrome panels, it can be done at this time. After acquisition and analysis, compare the median fluorescence intensity with the luminescence results. If you click on this LINK and scroll down to the bottom of the page, you will see an example of how results from OxyFLOW™ can be compared with those of HALO® for etoposide, a compound that produces double-stranded DNA damage.