Abstract
Enzyme-linked immuno spot (ELISPOT) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of ELISPOT assay is a formation of colored spots which appear at the sites of cells releasing cytokines, with each individual spot representing a single cytokine-releasing cell. We worked out a protocol to study oxidative stress in human peripheral blood lymphocytes by determining their potency to secrete IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha in response to acute treatment with hydrogen peroxide. We show that hydrogen peroxide-induced oxidative stress can cause a ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokines IL-8 and TNF-alpha. However, the number of cells secreting TH2 cytokines IL-4 and IL-5 in hydrogen peroxide-treated group did not change. It appears that oxidative stress may affect TH1–TH2 cytokine secretion balance which, in turn, may underlie developments of various pathological conditions. This protocol can be easily modified to study the effects of many other oxidative stress compounds.
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Acknowledgments
Christopher Hartnett for assistance with isolation of peripheral blood lymphocytes.
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Hagen, J., Houchins, J.P., Kalyuzhny, A.E. (2012). ELISPOT Assay as a Tool to Study Oxidative Stress in Lymphocytes. In: Kalyuzhny, A. (eds) Handbook of ELISPOT. Methods in Molecular Biology, vol 792. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-325-7_7
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DOI: https://doi.org/10.1007/978-1-61779-325-7_7
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