how to add stimulants and how many micrograms of each protein is appropriate

[Guest TANG]

Dear Christian
I am rather poor in English and I hope you can understand my question.
I immunized mice with 4 recombinant proteins and took spleens for elispot 2 weeks after the third immunization to detect IL-4 and γ-IFN, but I don't know how to add stimulants and how many micrograms of each protein is appropriate?

[Christian@mabtech.com]

Dear Tang, welcome to the forum!

When setting up ELISpot looking at antigen specific responses it is good to do some trial experiments where you titrate cells and antigen. These aspects are important:

- Make sure you run precoated ELISpot plates from Mabtech. You can ofcourse coat yourself but by running precoated plates you are guaranteed a well performing assay.

- Splenocytes should be isolated under optimal conditions and diluted in appropriate cell culture medium. We have a tutorial on our new website: https://www.mabtech.com/knowledge-hub/isolation-freezing-and-thawing-splenocytes

- Mix your antigens in cell culture media and add them to the ELISpot plate in titration series to determine optimal stimulation conditions. Most antigens work well at 2ug/ml but I would try 4ug/ml, 2ug/ml, 1ug/ml and 0.5ug/ml in order to determine the optimum. Set up these test wells in triplicates so that you get good statistical power.

- Titrate your splenocytes. Typically you run antigen ELISpot at 200-250k cells/well. However, some antigen can require more cells and sometimes responses are so strong that you get confluent spot formation at 200-250k. Thus, set up a titration where you test your antigens at 400k cells/well, 200k cells/well and 100k cells/well. 

- Cytokines such as IFNg often get good result after overnight incubation, but some antigens require 2 days of incubation to see clear antigen specific responses. This is especially true for TH2 cytokines such as IL-4. As a result, please evaluate in trial experiments plates that have been incubated 20h and 40h, compare the data. 

 

EDIT:

Here is the response in Chinese:

你好，Tang，欢迎来到我们的论坛！

 

在做ELISpot检测抗原特异性反应之前，最好做几次预实验来确定每孔细胞数和抗原浓度。下面这几点比较重要：

 

- 确保使用 Mabtech 的预包被 ELISpot 板。 当然可以选择自己包被，但使用预包被板的话可以保证预实验及后续实验的顺利。

 

- 应在优化的实验条件下分离脾细胞，并在适当的细胞培养基中稀释。 我们的新网站上有教程：

https://www.mabtech.com/knowledge-hub/isolation-freezing-and-thawing-splenocytes

 

- 在细胞培养基中以不同浓度稀释抗原，然后将它们加到 ELISpot 板中进行滴定，以确定最佳刺激条件。 大多数抗原在 2 µg/ml 的浓度效果很好，但我建议尝试 4 µg/ml、2 µg/ml、1 µg/ml 和 0.5 µg/ml 的梯度以确定最佳浓度。 每种条件做三个复孔，以便获得统计上有意义的结果。

 

- 滴定每孔脾细胞数。 通常情况下，抗原特异性T细胞 ELISpot应种每孔20到25万细胞。 然而，某些抗原可能需要更多的细胞；而有些抗原引起的反应非常剧烈，以至于每孔20到25万细胞会造成斑点融合。 因此在预实验测试抗原时，需通过每孔40万、20万、10万细胞来滴定。

 

- 不少细胞因子通常在孵育过夜后就可以得到良好结果，比如IFN-γ 。但某些抗原需要孵育两天才能看到明显的抗原特异性反应。 对于 TH2 细胞因子（如 IL-4）尤其如此。 因此，请在预实验中尝试孵育20和40小时来进行比较。

 

祝好！

克里斯蒂安

best,

Christian