UBC Theses and Dissertations
Raw 264.7 macrophage-mediated depletion of hydrogen peroxide in cell culture media Karastathis, Dimitrios Nestor
Macrophages are known to produce hydrogen peroxide that act both as part of the host defense system, and as secondary messenger. As hydrogen peroxide has powerful effects and macrophages, which are found at rough implant surfaces, must function in an environment containing it, it is of interest to know how hydrogen peroxide levels are regulated. The current study was conducted in order to determine whether RAW 264.7 macrophages were capable of depleting exogenously added hydrogen peroxide. Conditions for optimal detection of H₂O₂ using Amplex Red fluorescence were investigated under a variety of conditions. The ability of RAW 264.7 cells to produce and/or deplete extracellular hydrogen peroxide was determined when cell concentrations, times and incubation temperatures were varied. Other possible influences were also examined, including passages of cells in culture, H₂O₂ stimulation with LPS, and catalase inhibition via 3AT. Using Amplex Red to detect hydrogen peroxide, it was found that PBS and black welled microplates provided the most reliable conditions for detection. In addition, direct contact of Amplex Red with RAW 264.7 macrophages was found to interfere with the detection reagent’s ability to detect exogenous hydrogen peroxide. Most importantly, RAW 264.7 macrophages were found to deplete exogenous hydrogen peroxide, regardless of any previously treatment with LPS. This depletion was found to be time, cell concentration, temperature and passage number dependent. The use of a catalase inhibitor, 3AT, had no effect on reducing the ability of RAW 264.7 cells to deplete hydrogen peroxide from its surrounding environment, and was also found to lower the detection capacity of Amplex Red. The measurement of H₂O₂ produced by RAW 264.7 macrophages is difficult to determine, as our results using a specific H₂O₂ detection reagent demonstrated that the macrophages depleted any H₂O₂ in the media. The manner of depletion was found to be time, cell concentration, temperature, and passage number dependent. The anticipated stimulating effect of LPS was not observed, and the anticipated inhibitory effect of 3AT could not be detected, suggesting that macrophage depletion of exogenous hydrogen peroxide did not involve catalase.
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