Jump to content

Kajsa

Moderators
  • Content count

    10
  • Joined

  • Last visited

About Kajsa

  • Rank
    Member

Profile Information

  • Gender
    Not Telling

Recent Profile Visitors

332 profile views
  1. Thank you for your question! My short answer would be: No, I would not distinguish between the cells based on spot size. I think that by looking at the size you would get a hint on whether it is plasma cells or memory cells but I would not state it in the result section of your paper. Perhaps you could discuss it in the discussion section. WHY? To answer this I would go back to what we all know about plasma cells and memory cells: In the spleen you have the early reaction against antigens which forms plasmablasts (low affinity but antigen specific antibodies, possibly switched) and around 7 days post immunization, germinal centres are seen, generating plasma cells and memory cells (switched and with high affinity). Some plasma cells will subsequently migrate to the bone marrow where they become long lived plasma cells. So, in the bone marrow you will likely have plasma cells and not so many memory cells… but also many B cells under development, meaning they have more polyreactive and autoreactive BCRs. Will they respond to your antigen with antibody production? – Possibly? Furthermore, memory cells may also form plasma cells upon restimulation. Would you then be certain that your “plasma cells” found in the spleen three days post immunisation really are plasma cells and not restimulated memory cells? I don’t know. BUT THEN AGAIN… I thought I would also just mention the work of the Bryan Charleston lab. It might be of interest since they study plasma cells and memory cells after immunization using ELISpot BUT in cows so I don’t know how applicable their results are to you. Anyway, they argue that if you take PBMC from the immunized animal, the spontaneously formed spots are from plasma cells since they produce antibodies without further stimulation. If the cells are cultured for 6 days without stimulus they don’t see any spots but if the PBMC are stimulated with a mix of cytokines (IL2, IL10), anti-CD40 Ab and pokeweed mitogen they claim that they trigger the memory B cell pool, thereby all spots seen after 6 days in culture are memory B cell spots. The equivalent that we offer for mouse is the polyclonal activator R848 + IL2 in case you want to try The paper is: A quantitative assessment of primary and secondary immune responses in cattle using a B cell ELISPOT assay Lefevre et al. Vet. Res. (2009) 40:03. You were however thinking that you would just FACS them and see instead. I think that is a good idea, however maybe not instead. Is this your big finding that you will base your paper on? Do both. For inspiration: Dogan et al. Nat Immunol. 2009 Dec;10(12):1292-9. Multiple layers of B cell memory with different effector functions. Interesting for you is that at 3 days post antigen boost AID+ B cells, here memory cells, were transformed into plasmablasts. Gating for FACS: Kathryn A. Pape et al. Science. 2011 Mar 4; 331(6021): 1203–1207. Different B cell populations mediate early and late memory during an endogenous immune response. I normally do a simple Fas and GL7 staining for GC, pregated on B cells and use with a not B cells dump channel. With FACS you may see antigen specific plasma cells and memory B cell percentages (I recommend you enrich the B cells first (using e.g. MACS) to get the chance to even see them – make sure to recalculate the percentage to real numbers) but you can’t see their antibody producing capacity. You can always sort the cells and then plate them for ELISpot or FluoroSpot and check for a difference in the spot size to further straighten you discussion. If you think your treatment might affect the antibody production, do ELISpot and FluoroSpot in addition to FACS. If you think that the treatment only affects cell number – FACS might be enough. Good luck Kajsa
×