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Fluorospot and protein coating

Guest Laura P2023-06-30T09:19:02Z

Hello!

I have been performing triple (IgG/IgM/IgA) B-cell fluorospot to assess the number of antibody-producing cells in plasma cells isolated from immunized mice. I coated some wells with anti-Ig and some with the protein fragment used for immunization, in order to have the number of cells producing any Ig and specific Ig. This works usually well, but with one of the antigens I get very undefined, diffuse spots, only in the case of the wells coated with the protein and only for IgG. I attach a file showing this effect. What could be the cause of this? I thought it could be a coating problem, but the specific IgM and IgA spots are looking good. Would you recommend trying with reversed B-cell fluorospot in general, in order to avoid antigen-coating related issues?

Thank you!

Have a great day,

Laura

20230630 Fluorospot troubleshooting.pdf

Renata2023-06-30T11:48:10Z

Hi Laura,

It’s great to see that you have an antigen-specific response, but the IgG spots are indeed a little bit diffuse! The diffuse pattern may be due to the nature of the antigen itself, but it is often caused by a suboptimal antigen concentration used for coating. Have you tested several coating concentrations of this antigen? The concentration required may really depend on the antigen itself, so perhaps that is why it worked well with other antigens, but not so well with this one. I would recommend testing several higher concentrations to see if that improves your results.

Reversed B cell FluoroSpot is always an option too, but that comes with its own optimization requirements. I think it’s best to start by testing higher coating concentrations and take it from there.

In addition, have you included a PBS-coating control? Or coating with an irrelevant antigen? We recommend this to make sure that the IgM spots are not just polyreactive IgM, but are actually true antigen-specific IgM.

All the best,
Renata

Guest Laura P2023-06-30T13:12:08Z

Hi Renata,

Thank you very much for your response!

I have not tested different coating concentrations of the antigen, as we always have very limited amount of cells, but I plan to test higher concentrations. I will follow your advice and start with this then.

I did not include PBS-coating or irrelevant coating in this fluorospot (again, due to the limited material). I will include them in future experiments, when we get material from more mice at once.

Thanks again for your help!

Best,

Laura