22 April 2020 0 9K Report

Hello RG community,

I’m using reporter cell lines (NF-kB) and have a few technical questions:

  • For the pathway to function ie TNF-a activating NF-kb reporter, are endogenous cellular components vital/ used/ necessary, as in does the host cell provide things like IKKg, IkBa, p65 etc for the reporter to “work” of are these simply bypassed as the cassette is a yes no can this function / be activated YES or NO, the pathway itself as a whole is actually not utilised.
  • I have some supernatant which blocks activation of a cell line (ie HEK, THP1) as in cells + activation cytokine etc = IL-8 via ELISA can block with incubation with my SN, so the inflammation and / suppression pathways are there and functional but the same line is not giving us the same answer as the reporter pathway. I can activate, I can use a commercial chemical to supress but the supernatant only works to supress activation via ELISA not via the reporter. This doesn't make sense to me as we are looking at transcription (reporter, does the pathway work) v’s translation (actual output of a cytokine).

    • Any idea why I would see an ELISA work for suppression but not the reporter (same line)? I could see the reporter working but then maybe not the ELISA if the full pathway is abrogated etc but not the reverse and the reporter function is much earlier upstream than translation to a cytokine output.

    Thanks for any idea/ suggestions/ discussion

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