Abstract
The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells will emerge from the bone marrow. Thus all B cells that were exposed to the antigen and still alive after at least 4 weeks (8 weeks or more is ideal), are memory B cells. Splenic B cells from B1-8 mice were prepared with an EasySep Mouse B Cell Enrichment Kit according to the manufacturer’s protocol. Single-cell suspensions were transferred intravenously into tail veins of recipient mice. Approximately 1 million NP+ B cells were transferred per mouse. Approximately 12-24 h after transfer, mice were immunized intra-peritoneally with 50 µg of NP-CGG precipitated in alum.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Flow cytometry of splenic cells from AM14-Tg x Vκ8R-KI recipient mice given NP-specific B cells and immunized with NP-CGG in alum, assessed 8 weeks later. Number adjacent to outlined area indicates percent CD19+NP+ antigen-specific B cells among live cells. (from Zuccarino-Catania et al., 2014)
Figure 2. Flow cytometry of splenic B cells from AM14-Tg x Vκ8R-KI mice immunized with NP-CGG in alum without transfer of NP-specific B cells, assessed 8 weeks later as in Figure 1. (from Zuccarino-Catania et al., 2014)
Notes
Recipes
Acknowledgments
This protocol was developed or modified in Dr. Mark Shlomchik’s laboratory at Yale University. Supported by the National Institutes of Health (R01-AI46303 to M.J.S) and NSF Graduate Research fellowships (G.V.Z.-C.). This protocol was adapted from Tomayko et al. (2010) and Sweet et al. (2013).
References
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