I'm trying to develop a stable model for B cell memory. for that, i kept mice for 2 months (~60 days) after primary immunization with OVA ~80ug OVA/Alum , and gave a boost next, and 6-7 days later, i sacrificed the mice and checked the spleenocytes for MBCs. Spleens of OVA group were much enlarged compared to the control group. However, im quite disappointed with the Flowcytometry results. as they show no difference between Control and OVA group. both have developed Memory b cells, and have minor to no difference in the percentage. The FC panel i used is "PI- B220+ CD38+GL7- IgG or IgM + ".
i did check for T cells, in that case i did notice a difference in CD44+ CD62- population (increased) and CD44+Cd62+ population (decreased) in OVA group, which is suggestive of an immune response activation in OVA vs Control group.
Further, i also noticed that 3/8 Mice (2 female and 1 male) died after Boost (secondary immunization.
How can i improve this protocol. Any suggestions/ references are welcome.
Thank You.
A few things to consider:
1. What was the route of injection? Memory cells (for the antigen) reside in DRAINING lymph nodes and eventually bone marrow. Spleen is not the best place to look for them unless you do IV injections.
2. Try different time intervals after the booster
3. OVA is not a highly immunogenic antigen. You might try KLH.
Ellen S Vitetta Thank you for your response,
i will try the above mentioned suggestions.
route of injection was ip.
i count the absolute number and found significant difference between groups. however, i also noticed that almost all the b cells i detected were also CD38+ and ~97% of them were even IgG+ as well. GL7+ was a very minor population. could it be that after 2 months the GL7+ has disappeared? but how come they all become memory, i.e CD38+ igG+ ?