FluoroSpot and iSpot FAQ

 

 
 

1. I plan to start doing fluorescent-based EliSpot assays but I want to be able to handle traditional ELISpots as well. Is this possible with any one of your machines?

2. Is cross-talk between fluorochromes a problem with the iSpot Reader System?

3. How many different fluorescent dyes can be analysed with the iSpot system?

4. Which FluoroSpot assays do you recommend?

5. Which technique is used to detect fluorescent spots in a FluoroSpot assay?

6. How long does it take to analyse a FluoroSpot plate?

7. Can the iSpot detect spots which contain a large amount of IFN-γ and very little IL-2 and vice versa?

8. Which filter set comes with the iSpot reader and is it possible to have other filter sets?

 
 

 
 

1. I plan to start doing fluorescent-based EliSpot assays but I want to be able to handle traditional ELISpots as well. Is this possible with any one of your machines?

Yes, the new AID iSpot Reader can cope with both types of applications. One mouse click in the software and one single switch in the hardware and you are ready for the future in EliSpot.

 

2. Is cross-talk between fluorochromes a problem with the iSpot Reader System?

Crosstalk is an often observed and unwanted phenomenon that occurs when more than one fluorochrome is analysed in the same assay. It is particularly problematic when dyes with overlapping excitation spectra are used, further so when the intensity of one of the analysed cytokines dominates over the other. The AID iSpot machine is equipped with highly specific filter sets, which avoid cross-talk, even when the excitation spectra of the involved fluorochromes are quite close. For the FluoroSpot assays available from Mabtech, cross-talk is not a problem at all since the excitation spectra of the fluorochromes used (FITC and Cy3) are adequately separated.

 

3. How many different fluorescent dyes can be analysed with the iSpot system?

With the highly flexible custom-made 7&1-filter cube it is, in theory, possible to analyse up to 21 different dyes (cytokines). However, FluoroSpot assays are pretty much at the beginning of development and the commercially available kits are so far only using two different dyes. Processing with a high resolution system may take slightly longer. The AID Reader is still the fastest EliSpot machine on the market.

 

4. Which FluoroSpot assays do you recommend?

AID recommends the FluoroSpot assays from Mabtech Sweden (www.mabtech.com). Their kits are well tested in the iSpot Reader.

 

5. Which technique is used to detect fluorescent spots in a FluoroSpot assay?

During the last three years AID has put a great deal of effort into FluoroSpot technology and has developed a multiple filter based system which, in combination with the sophisticated image overlay technology, has several significant advantages over the universal filter solution used by comparable machines. The AID iSpot solution will identify double or triple stained spots by their position in the well rather than by their color. This will define double-stained spots even if the signal of one of the detected cytokines is weak compared to the other.

 

6. How long does it take to analyse a FluoroSpot plate?

Compared to the complete analysis of an enzymatic plate, which usually takes less than 2 min, the imaging and counting is a little more time-consuming. However, a typical 96 well IFN-γ/Il-2 assay will be ready in around 5-6 minutes. This is because the reader has to record at least two different images per well in a FluoroSpot assay.

 

7. Can the iSpot detect spots which contain a large amount of IFN-γ and very little IL-2 and vice versa?
Yes, this is possible without any problem since the iSpot reader works with highly specific filters and with different images, which are overlayed. The software will find the double stained spots based on their position and therefore it is not a problem if the cytokines analysed are secreted in different amounts.

 

8. Which filter set comes with the iSpot reader and is it possible to have other filter sets?
The AID iSpot reader is equipped with specific filters for Cy3 and FITC to analyse commercially available FluoroSpot assays based on anti-cytokine reagents containing these fluorochromes (e.g Mabtech human IFN-γ/Il-2 FluoroSpot assays). The instrument can be supplied with different filters on demand.

-

 
 
 

 

 

     
 

For information about our other diagnostic products for example Immobilized DNA probes, Immunoblots or Immunofluorescence, please visit our website:

 

     
  www.aid-diagnostika.com  
         
  © AID GmbH 2000-2015 - All rights reserved