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Irregular spot-formation in ELISpot, why?

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Dear Mabtech Customer Service Team!

We are routinely using your Mabtech IFNg ELISpot kits (#3420-2APT-2 or #3420-2AST-2) for our experiments. Recently, we observed some strange looking spots we don`t have an explanation for, maybe you can provide me with your expert opinion on this?


Experiment set-up: purified monocytes are matured to dendritic cells; these are then loaded with peptides and incubated together with purified CD8+ T cells on the IFNg ELISpot plates. Usually we get distinct spots; however, in the last experiment we observed some kind of clustering of cells (see the attached powerpoint), and we don`t know how to interpret these data.


Maybe you can help us? Do you know how such clustered spots might be caused?


Thank you very much and best regards,


Examples of ELISPOT trails.pptx

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Dear Customer,



The phenomenon of irregular spot-formation is quite common in ELISpot especially when doing longer incubation times. The explanation that is usually put forward is that the IFN-g secreting T-cells have moved on the membrane during incubation and essentially formed “trails” of secreted cytokine on the PVDF membrane.


Cells are quite motile during incubation, in particular when activated. This motility is different in donor-to-donor and may very well depend on the specific cytokine environment that is established within the cell culture. Important to point out is that this cytokine environment is very much influenced by the dendritic cell populations as well.


When I get these sort of trails I don’t change anything in my analysis. I let my ELISpot reader count the spots using my standard settings.


Looking at your wells I kind of notice that your camera seems to be a bit out of focus. I presume you are using an AID reader, right? We at Mabtech have done this Youtube tutorial for how to setup and use an AID reader in an optimal way. Here is the link: https://www.youtube.com/watch?v=SPuFjr44U-I


The video is long but it is worth it J





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