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Small Spot Size and High Background

Guest Sophia

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Hi Mabtech,


I am currently testing the response of cytokine secreting t cells after rabies vaccination by stimulated PBMC using peptide pool. I used FluoroSpot Plus: Human IFN-y/IL-4 kit. The result I got seems to be very odd with the size of spot in antigen stimulated condition since almost all of the spot are small, which is very hard for me to differentiate them with the background. Also, the amount of spot are very little than what I expected to see because I expected to see higher amount of cell in day 14 and 6 months time point compare to day 0. I would also want to know what could be the reason the high background in antigen stimulated condition. One more thing, in FITC, there are some spot that have a weird looking shape, I think those are not t cells, so I excluded them. 


Best Regards,




Cytokine Secreting T Cells.pdf Cytokine Secreting T Cells.pdf

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Dear Sophia,

Technically your spots look very good so I am confident you have done the assay in the right way. However, there are some question marks I have relating to your experimental setup:

1. Are you studing a new form of rabies vaccine that you have developed yourself? There is very little response in IFNg at 6months, which could reflect that the vaccine is not working well. Can you give me some background?

2. The positive control wells, what stimuli did you use here? Was this antiCD3 at 300k cells/well?

3. The concentration of the peptides is rather high at 10ug/ml. How many peptides is this? Is it the concentration of each peptide or combined?

4. Did you assess the viability of the cells after thawing them? The lack of strong positive control spots makes me suspicious that they are not feeling well.



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Hi Christian,


Thank you for your immediate response.

Regarding your 1st question, the PBMC are from patient who received rabies post-exposure prophylaxis endorsed by WHO. So I wanted to see if those individual have any T cells response after 6M of vaccination. However for other sample which I did not include in the pdf file, the IFN-g are pretty high for antigen stimulated well, but also high for negative control, which is very odd.

To answer your 2nd question, yes I did use anti CD3 at 300K cells/well.

To the 3rd question, this is the custom peptide, which does not mean I add more than one peptide per well, it is peptide pool covering rabies glycoprotein which is in the same tube, so the total concentration for each experiment was 10ug/ml or 1ug per well.

To the 4th question, I did check the viability. I only included those with viability higher than 70%.





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The antiCD3 response at 300K is way too low for IFNg and IL-4. This is a clear indication that cell viability is too low giving suboptimal results. 

In humans, Merck did this landmark study around 2006 and found that cell viability needed to be above 84%, otherwise you dramatically lost antigen specific responses in ELISpot. For really weak antigen responses above 90% is recommended. 

We tend to use peptides at 1-2ug/ml. But if this pool covers a range of peptide segments it can ofcourse quickly amount to 10ug/ml. There is a strange background in IL-4 in the wells stimualted with peptide, which do not show up in IFNg. This seems to indicate some sort of selective issue which can be controlled for by incubating peptide in cell culture medium but without addition of any cells. If the background is still there you would know it is not cell related. 

Getting high viability in thawed samples of PBMC requires a whole chain of correct cell handling and freezing. We have written guides about this on our updated website:



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