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FluoroSpot questions


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I’ve been reading about FluoroSpot and have a few questions:


1.       What are the advantages of this method compared to intracellular FACS?

2.       I have some experience with ELISpot, would this be of use? The techniques seem rather similar…

3.       What kind of reader is required?





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Emma, welcome to the Mabtech forum! Let me try to answer these questions for you:



FluoroSpot and intracellular cytokine staining (ICS) in combination with flow cytometry both share the nice property of being able to detect cytokines at the single cell level. Nonetheless, FluoroSpot is generally acknowledged as being a more sensitive technique and better suited for high throughput screening. But apart from this, other important differences exist as well:


First, the FluoroSpot technique detects cytokines that have been actively secreted by the producing cells (i.e. biologically relevant cytokines). By contrast, ICS only detect cytokines that have accumulated inside of cells usually in the presence of a protein transport inhibitor such as Brefeldin A. As a result, a positive population of cytokine stained cells in flow cytometry does not necessarily translate into cytokines that would have been destined for cytokine release. In FluoroSpot this is not an issue.


Second, FluoroSpot is an “accumulative” method that captures cytokines during the entire period of incubation (24h, 48 or 72h for example). By contrast, intracellular cytokines in ICS are only captured during a limited “window” of Brefeldin A incubation, usually lasting 4-6h and often preceded by a standardized period of pre-activation (many times 16-20h). Thus, any cytokines produced prior to the addition of this Brefeldin A will not be detected using ICS. You run into the risk of simply missing important populations of cytokine producing cells, which is not the case in FluoroSpot.


Third, flow cytometry has the ability to combine the detection of intracellular cytokines with that of cell surface markers. FluoroSpot does not have this luxury. Consequently, in order to analyze a particular population, cells will have to be sorted prior to being added into the FluoroSpot wells using either a cell sorter or magnetic cell separation. In any case, this involves a much more complicated workflow compared to just staining for a couple of CD markers in FACS.  


As of now, flow is able to analyze more cytokines simultaneously compared to FluoroSpot. On the other hand, multi-color flow cytometry is technically challenging to perform and are many times tide to a core-facility. FluoroSpot has been designed to be handled by any lab, anywhere.




Yes, you are absolutely right! Not much difference.


The FluoroSpot assay is the natural advancement of the ELISpot assay that uses fluorescent detection instead of colorimetric. By being able to use fluorescence, the possibility of detecting two analytes simultaneously opens up a lot of interesting possibilities. Other than that, the two methods are identical.




A so-called FluoroSpot reader is needed to analyze these plates at a high-throughput. Most reader companies nowadays equip their ELISpot readers with Fluorescence filters to make their machines “FluoroSpot ready”.  



I hope these answers helps!


If anyone else reading this forum does not agree with my opinions, you are much welcome to register and tell me where I am wrong.

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  • 2 years later...

Hi Emma and Christian,


I don't disagree with you, Christian. But coming originally from a research group where flow cytometry was the go-to technique, I made the below list to convince myself of why the heck I would run a FluoroSpot assay intstead of just doing ICS and flow cytometry. The list contain most of what you bring up above, but a few bullet points haven't been mentioned by you. I thought it could add some to your discussion. Enjoy.


Why FluoroSpot and not intracellular cytokine staining and Flow cytometry?

  • More sensitive – capture analytes throughout the stimulation making it possible to detect early as well as late producers.
  • More robust – suitable for multicentre trials.
  • Less cells needed to get a reliable result.
  • Physiologically relevant secretion – not clogged up production.
  • Kinetics of cytokine release less of a problem – Monensin and Brefeldin A affect cytokine stainings differently.
  • Viable cells after assay – possible to continue culturing the cells.
  • Although no surface markers – what really matters is the functional cytokine profile.
  • Detection of antibody secreting cells difficult with Flow cytometry, but straight-forward with FluoroSpot and ELISpot.
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