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Guest Yestin Yang

Question from China: IFNg/GzB FluoroSpot

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Guest Yestin Yang
We are doing the first experiment for IFN-g detecting by the new AID machine. Then we will use the IFN-g/GrB fluorospot kit to evaluate the activity of cytotoxic T lymphocytes before and after the cancer patients received the antitumor vaccines. Some researches concluded that IFN-g ELIspot assay alone was not sufficient to detect the difference between the prevaccination and postvaccination samples.
 
Could you provied me some references and experience for IFN-g/GrB using?
And can you share your experience when you use the cryopreserved cells in ELIsport assay?
 
Another question, do you have the catologe No. of the empty plate frame for transfer of strips?
 

 

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Dear Yestin Yang,

 

 

Your interest in our IFN-g/GzB FluoroSpot kit (https://www.mabtech.com/products/fs-0110-2_human-ifn-γ-granzyme-b-fluorospot-kit#tabs-min-1) sounds very reasonable from a scientific point of view. By combining the cellular secretion of both IFN-g and GzB, a population of "true cytotoxic" T-cells can be identified, and in principle better correlate to vaccine efficacy.

 

Granzyme B is in general a more "difficult" analyte to investigate compared to Interferon-gamma. Spots are less distinct and many donors will have high backgrounds from unstimulated PBMC. However, by using FluoroSpot and looking at the number of double secreting cells, GzB becomes a very valuable complement to the information and stimulation index provided by IFN-g. In our hands the assay works very well, especially if you incubate your cells for 48hours as Granzyme B tend to look better at with longer incubation times.   

 

There are currently no publications utilizing the IFN-g/GzB FluoroSpot assay. We hope this is going to change in the coming years. Nonetheless, to demonstrate how this assay looks in our hands I have attached a powerpoint showing you a typical result using 250,000 PBMC/well that have been stimulated for 48h in the absence or presence of the CEF peptide pool (https://www.mabtech.com/products/3615-1_cef-peptide-pool#tabs-min-1). Despite a high background in medium ctrl for the GzB, the number of double secreting cells has an excellent stimulation index of 31 spots to 1! 

 

 

Analyzing cryopreserved PBMC in ELISpot works well provided that the cells have been frozen and stored in a suitable manner. Studies have shown that once viability drops below 80%, consistency in ELISpot data goes down significantly. So you want to be in the range of 80-99% viablity for good results. The higher, the better, naturally. At Mabtech, the majority of quality control and research & development is done using cryopreserved PBMC. You can read more about cell handling in our tutorials & Guidelines (https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/cell-handling).

 

 

The empty plate frame used for the ELISpot strip plate is not sold as a separate item. However, we should be able to send you one next time you order. Just ask your distributor to contact us in advance to sort out the details.

 

 

/Christian

Fluorospot_IFNg_GzB.pptx

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Guest Yestin Yang

Dear Yestin Yang,

 

 

Your interest in our IFN-g/GzB FluoroSpot kit (https://www.mabtech.com/products/fs-0110-2_human-ifn-γ-granzyme-b-fluorospot-kit#tabs-min-1) sounds very reasonable from a scientific point of view. By combining the cellular secretion of both IFN-g and GzB, a population of "true cytotoxic" T-cells can be identified, and in principle better correlate to vaccine efficacy.

 

Granzyme B is in general a more "difficult" analyte to investigate compared to Interferon-gamma. Spots are less distinct and many donors will have high backgrounds from unstimulated PBMC. However, by using FluoroSpot and looking at the number of double secreting cells, GzB becomes a very valuable complement to the information and stimulation index provided by IFN-g. In our hands the assay works very well, especially if you incubate your cells for 48hours as Granzyme B tend to look better at with longer incubation times.   

 

There are currently no publications utilizing the IFN-g/GzB FluoroSpot assay. We hope this is going to change in the coming years. Nonetheless, to demonstrate how this assay looks in our hands I have attached a powerpoint showing you a typical result using 250,000 PBMC/well that have been stimulated for 48h in the absence or presence of the CEF peptide pool (https://www.mabtech.com/products/3615-1_cef-peptide-pool#tabs-min-1). Despite a high background in medium ctrl for the GzB, the number of double secreting cells has an excellent stimulation index of 31 spots to 1! 

 

 

Analyzing cryopreserved PBMC in ELISpot works well provided that the cells have been frozen and stored in a suitable manner. Studies have shown that once viability drops below 80%, consistency in ELISpot data goes down significantly. So you want to be in the range of 80-99% viablity for good results. The higher, the better, naturally. At Mabtech, the majority of quality control and research & development is done using cryopreserved PBMC. You can read more about cell handling in our tutorials & Guidelines (https://www.mabtech.com/knowledge-center/tutorials-and-guidelines/cell-handling).

 

 

The empty plate frame used for the ELISpot strip plate is not sold as a separate item. However, we should be able to send you one next time you order. Just ask your distributor to contact us in advance to sort out the details.

 

 

/Christian

Thanks so much for your expert advice on these questions.We will test the  IFN-g/GzB FluoroSpot kit later.

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As Christian wrote, there were no references to IFNg/GzB FluoroSpot back in 2014. But already in already in April 2015, Weinberg et al published this paper where the authors utilized IFNg/GzB FluoroSpot to evaluate T cell responses in a vaccine context: https://www.ncbi.nlm.nih.gov/pubmed/25874544

 

The paper is published in the open access journal PLOS ONE, so anyone can download it. Happy reading! 

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