Dear all,
I'm going to quantify memory B cells using ELISPOT assay. I will use splenocytes from immunized C57BL/6 mice.
Could you recommend any possitive control for this?
Thank you very much!
Dear Quyen,
are you going to perform antigen-specific Elispot by coating the antigen? Then, you could just coat anti-Ig for your positive control. Or are you looking for an unspecific B cell stimulus to induce antibody secretion in your positive control. Here, LPS might do the job. I also attached a paper describing two different ways of doing an B cell Elispot.
Good luck,
Michael
http://www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900407
I agree with Michael and would add that if you want to evaluate the specific memory B cells, You can also use the memory B cell specific for the tetanus toxin because almost everyone is vaccinated and should have enough of these cells
Good point Bourema,
but only valid for human samples. Quyen is working with murine samples. I would be suprised to see tetanus responses in these animals.
Best,
Michael
Thank you very much for your comments, Michael and Bourema.
I evaluate specific memory B cells and i try to do in 2 ways as you known. Coating with a specific antigen and anti Ig. I woul like to know about stimuli actually. From a paper, I found out that R848 +IL2 are used to induce antibody secretion. But my lab now doesnt have R848. LPS is used as TLR4 argonist and i'm not pretty sure about using it to induce antibody production. So I tried PHA 5ug/ml + IL2 20ng/ml and waiting for the results now :)
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