High background level and Repeatability

[Xian Yang]

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.

[Xian Yang]

I'm sorry, it looks like the names of these images won't be uploaded. The six pictures are, in order, PMA+Placebo-immunized, PMA+Vaccine-immunized, Stimulsted+Vaccine-immunized, Stimulsted+Placebo-immunized, Unstimulsted+Placebo-immunized, Unstimulsted+Vaccine-immunized.

[Christian@mabtech.com]

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. 

[Xian Yang]

Thank you so much for the reply!

Before me, my colleagues waited two weeks or nine weeks, and eventually they chose to remove the lung after one week. As you said, although the background is high, there is still a considerable antigen induced response.