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Team:UCSF/Protocols/Assays
From 2010.igem.org
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| - | <h3 style="font-weight:bold;">Illustration of the | + | <h3 style="font-weight:bold;">Illustration of the reporter assays used for measuring killer cell activation</h3><br> |
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| + | <p> We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. This assay format would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells We looked at two reporter readout: one was activation of the NFAT (nuclear factor of activated T cells) promoter driving GFP expression (by FACS), the other was IL-2 expression/secretion which is downstream of NFAT activation (by ELISA).</p> | ||
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Latest revision as of 21:28, 27 October 2010
Illustration of the reporter assays used for measuring killer cell activation
We made many devices for assay (we had a lot of ideas :) ), but unfortunately we ran into a problem with our idea to use cytotoxicity assays to measure the killing response. The problem was that the transient transfection/expression of our devices by electroporation was too low to see responses (<20% efficiency). The large amount of untransfected cells dominated the response. In brainstorming with our advisors we decided to use/substitute T cell activation assays as a reporter of potential killing response as they should be correlated. This assay format would allow us to look at transient transfected cell responses without a lot of background response from untransfected cells We looked at two reporter readout: one was activation of the NFAT (nuclear factor of activated T cells) promoter driving GFP expression (by FACS), the other was IL-2 expression/secretion which is downstream of NFAT activation (by ELISA).
