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Protocols  >  Laboratory protocol  >  IL-12p70 blocking assay

IL-12p70 blocking assay

Laboratory protocol

IL-12p70 inhibition by cytochalasin D
DCs were exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C. After washing with cold PBS, DCs were stimulated with S. cerevisiae yeast cells or spores at a DC:stimuli ratio of 4:1 for 24 hours. IL-12p70 production was assessed by ELISA.

IL-12p70 blocking assay
In order to assess the importance of IL-12p70 in balancing Th1/Th17 response two different experiments were performed.
In one case, DCs were stimulated for 8 hours with live spores of S. cerevisiae and C. albicans hyphae at a stimuli:DC ratio of 4:1 in the presence of different concentration of human recombinant IL12p70 (0, 1, 10 and 100 ng/ml).
In the second experiment, DCs were pre-incubated with different concentrations (0, 0.1, 1, 10, 100 microg/ml) of a monoclonal anti-human IL-12 antibody for 2 hours, then stimulated for 8 hours with live S. cerevisiae cells in a stimuli:DC ratio of 4:1.
The ability of these stimulated DCs to drive CD4+ T cells polarization towards a Th1 response was assessed by measuring of IFNgamma and IL-17 in T cells 5-day culture supernatants.

Protocol Steps:
  • IL-12p70 inhibition by cytochalasin D DCs are exposed to cytochalasin D (10 microg/ml, TebuBio) for 30 minutes at 4°C. After washing with cold PBS, DCs are stimulated with S. cerevisiae yeast cells or spores at a DC:stimuli ratio of 4:1 for 24 hours. IL-12p70 production is assessed by ELISA.
  • IL-12p70 blocking assay In order to assess the importance of IL-12p70 in balancing Th1/Th17 response two different experiments are performed.
    In one case, DCs are stimulated for 8 hours with live spores of S. cerevisiae and C. albicans hyphae at a stimuli:DC ratio of 4:1 in the presence of different concentration of human recombinant IL12p70 (0, 1, 10 and 100 ng/ml).
    In the second experiment, DCs are pre-incubated with different concentrations (0, 0.1, 1, 10, 100 microg/ml) of a monoclonal anti-human IL-12 antibody for 2 hours, then stimulated for 8 hours with live S. cerevisiae cells in a stimuli:DC ratio of 4:1.
    The ability of these stimulated DCs to drive CD4+ T cells polarization towards a Th1 response is assessed by measuring of IFNgamma and IL-17 in T cells 5-day culture supernatants.

IL-12p70 blocking assay Graph

created over 8 years ago (21 January 2010)    last modified over 6 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]