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High background level and Repeatability

Xian Yang

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Hello, my English is bad, please be considerate. Thanks!
I've been using the 3321-4AST for five months and have been having a problem with the high background of the unirritated well. Recently I've compiled past results and found that there has been a phenomenon that the background of the vaccine-immunized mice is significantly higher than that of the placebo-immunized mice. This has caused a lot of trouble for my experiment. I am new to immunology and have not found a good solution. I hope to get your help. 
Seven days after the immunization, I removed the lungs of the mice, treated them with red blood cell lysate, and isolated the lung lymphocytes of the mice using procoll. For the culture, I used RPMI 1640 medium with 10% fetal bovine serum at 200,000 cells per well. In each trial, the vaccine-immunized mice had a higher background than the placebo-immunized mice, leading to fatal false negatives. Do you have any good suggestions for improvement? 
I guess it may be because after immunization, there is some IFN γ in the differentiated lymphocytes of mice, which leads to the release of IFN γ even without any stimulation during the culture process. But I don't know whether IFN γ must be produced and released immediately, and whether IFN γ will be stored in the cytoplasm of differentiated lymphocytes? I have just come into contact with the knowledge of immunology, and I have not found the exact relevant literature. Do you know any knowledge in this regard? 
Besides, I found that the intra- and inter-experiment reproducibility is very poor, which makes the experimental results unconvincing. Do you have any suggestions for improving the experimental reproducibility?

Looking forward to your reply, thank you very much.







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Dear Xian,

It is usually recommended to wait 2-3 weeks following last injection before removing the spleen from mice. Otherwise you can have hyperimmunization and in vivo activated T-cells causing background in your ELISpot. 

You have here removed the lung after 7 days which might be abit early. At the same time, tissue derived T-cells tend to have higher backgrounds compared to blood/spleen derived T-cells. 

When looking at you wells I can see that there is some background in the last photo (unstim+vaccine immunized). However, the stimulated well goes up quite a lot, right? The ctrl well has almost no response at all, right?

Even if you have background you seem to have a considerable antigen induced response. 

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