ELISpot Assay as a Tool to Study Oxidative Stress in Peripheral Blood Mononuclear Cells

  • Jodi Hagen
  • Jeffrey P. Houchins
  • Alexander E. KalyuzhnyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 682)


Enzyme-Linked Immuno Spot (ELISpot) assay is widely used for vaccine development, cancer and AIDS research, and autoimmune disease studies. The output of an 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 have shown that hydrogen peroxide-induced oxidative stress was causing ∼twofold decrease in the number of lymphocytes secreting the TH1 cytokines IFN-gamma and IL-2, as well as chemokine IL-8 and cytokine TNF alpha. However, the number of cells secreting TH2 cytokines IL-4 and IL-5 in hydrogen peroxide-treated group did not change. Our ELISpot data indicate that oxidative stress may affect TH1-TH2 cytokine secretion balance which, in turn, may underlie developments of various pathological conditions. We adopted ELISpot assay for studying oxidative stress in human peripheral blood lymphocytes by analyzing the acute effect of hydrogen peroxide treatment on the frequency of cells secreting IFN-gamma, IL-2, IL-4, IL-5, IL-8, and TNF-alpha.

Key words

ELISpot Oxidative stress Peripheral blood mononuclear cells PBMCs IFN gamma TNF alpha IL-2 IL-8 TH1 and TH2 cytokines Reactive oxygen species ROS 



Christopher Hartnett for assistance with the isolation of peripheral blood lymphocytes.


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Jodi Hagen
    • 1
  • Jeffrey P. Houchins
    • 1
  • Alexander E. Kalyuzhny
    • 1
    Email author
  1. 1.R&D Systems, IncMinneapolisUSA

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