I plan to do an immunization and look at memory B cells among other B cell subsets. I will be doing a flow cytometric analysis (I will use cell surface markers and also look at antigen binding using a secondary detecting antibody) and I'm debating whether doing an ELISPOT in addition to this is beneficial and if so what information I can gain from it that the flow cytometric analysis will not give me.
Antigen-specific memory B cells are very rare, 5000-50000 in a whole spleen (= less than 0.1%). Therefore, flow cytometric identification of memory B cells is usually very difficult. ELISpot van help confirming the presence of memory B cells, but you have to be sure that the cells you plate out do not contain plasmacells/plasmablasts.
Elispot is a "functiona" assay as it allows to monitor not only the number of a given cell population (in your case memory B cells) but it will also allow to learn whether they are functional (through the production of a given cytiokine for example). Antigen-specific Memory B cells will of course be functional but since you will be getting small volumes of blood from your Imm. you may want to make sure that they are indeed the poulation you are looking to isolate...hope not too confusing for you
Taking in consideration the answer of prof. Suurmond, you could use FACS to get rid of the confunding B cell populations (plasma cells) and then seed the remaining cells on plates for ELISpot analysis.
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