Analysis of Germinal Center Reaction in Competitive Bone Marrow Chimeric Rag2-/-γc-/-Mice.

Germinal center (GC) reaction is crucial for the generation of high-affinity antibodies against any infection. To address the role of specific genes in GC, knockout mouse models are generally used. However, since GC is a multicellular event, complete knockout models cannot pinpoint the cell-intrinsic effect of certain genes in GC reaction. Here, we describe a detailed protocol for the analysis of GC in competitive bone marrow (BM) chimeric mice generated by transplanting 1:1 mixture of CD45.1 and CD45.2 positive donor bone marrow (BM) cells in immunodeficient Rag2-/-γc-/- mice as recipient host. We describe the method of immunization using sheep red blood cells (SRBC), detection of successful immunization by the antigen-specific antibody titer in the serum of the immunized mice and finally assessing the GC reaction in the spleen of the immunized mice by flow cytometry 10 days post-immunization. When the competitive chimera is made with wild type (WT) and a knockout (KO) donor BM, this method is suitable to address the cell type-specific (e.g., B-cell specific) role of the KO gene in GC.