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Patchy wells?

  • GL
    Guest Lai2018-12-27T19:10:55Z

    Hi All,

    How are you! I'm a postdoc in cancer biology field. Recently, I just started to optimise our ELISpot protocol for IFNg. I seemed to have the protocol nearly worked, but my recent experiment appeared that there are few wells (attached image) have very ugly patchy colour... I was not very sure what to cause it and was wondering if anyone can help me with it? After reading a few discussion here, would that be possible that it is caused by the well leakage or possibly rewetting the plate after drying?

    Thank you so much. Any suggestion would be appreciated.

    Bests,

    Lai

    ELISpot question.png

  • C
    Christian@mabtech.com2018-12-27T19:15:41Z
    Admin

    Dear Lai,

    Interesting case you got there. I dont have experience myself seeing this exact artefact but I do have a few guesses. But before I start posting educated guesses here are some questions for you:

    - What kind of cells are you running here? Is it human PBMC or mouse splenocytes? Are there a lot of red blood cells in your cell isolation?

    - You seem to be using Fast Red substrate, or am i incorrect?

  • GL
    Guest Lai2018-12-27T20:06:33Z

    Dear Christian,

    Thank you for the quick reply.

    The cells I used to run plates are mouse whole splenocytes. I have performed the ACK lysis on those samples so there shouldn't be too much RBC left and the cell pellet I saw after ACK lysis is white without too many red contaminations.

    The substrate I was using is AEC, which is a HRP substrate.

    Thank you so much

    Bests,

    Lai

  • C
    Christian@mabtech.com2018-12-27T20:52:21Z
    Admin

    I have one main avenue of thinking: there could be an issue with the substrate. The patches look artificial in nature and you have other ELISpot wells without any problems that are under the same condition, right? Could there be precipitation formation in the AEC? Do you filtrate this substrate prior to use?

    At Mabtech we recommend ALP in combination with our BCIP/NBT Plus substrate. Nevertheless, we recommend to filtrate the substrate prior to adding it to the wells in order minimize precipitation. You should contact us and ask us for a sample! Do a head-to-head against you current setup.

    //Christian