title: ELISPOT assay as a tool to study oxidative stress in lymphocytes.
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.
authors.affiliation: R&D Systems, Inc., Minneapolis, MN, USA.
authors.forename: Jodi
authors.initials: J
authors.lastname: Hagen
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Houchins
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Kalyuzhny
mesh_terms: D016207:Cytokines / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D058501:Enzyme-Linked Immunospot Assay; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000276:immunology
chemical_list: D016207:Cytokines; D006861:Hydrogen Peroxide