B cells contribute critically to an effective immune response by producing antigen-specific antibodies. are constructions that either activate B cells inside a polyclonal manner or antigens with repetitive motifs that induce B cell activation and antibody secretion without the need for T cell help (1). Thymus dependent antigens are typically proteins that induce a full immune response only after triggered B cells have received additional co-stimulatory signals from additional cell types such CX-5461 inhibition as T cells. After triggered B cells receive T cell help, they CX-5461 inhibition initiate germinal center (GC) formation (2). In the GC, antibody diversification happens Ctsk through somatic hypermutation, affinity centered clonal selection and growth (3). Furthermore, through immunoglobulin class switch recombination, the weighty chain of the produced antibodies and thus their function can change (4). The final output of the germinal center is the differentiation of GC B cells to plasma cells, which secrete large quantities of antibodies, and memory space B cells, which mediate sustained safety against previously experienced pathogens. These processes are crucial for an effective immune response and so far considerable effort has been put into elucidating how B cell activation, clonal growth, antibody secretion and B cell differentiation are regulated. Important tools with this effort possess included model antigens consisting of hapten-conjugated proteins (e.g. 4-Hydroxy-3-nitrophenylacetyl (NP)- chicken gamma globulin (NP-CGG)) and sheep reddish blood cells (SRBC). Unlike hapten- carrier conjugates, SRBC do not require the presence of an adjuvant CX-5461 inhibition to induce a strong GC response. Although SRBC are well suited to study GC formation and plasma cell differentiation, it has been difficult to analyze levels of SRBC-specific antibodies in serum from immunized mice. Protocols to analyze levels of SRBC-specific antibodies by ELISA are available; however, due to the difficulty and potential technical difficulties of these protocols, determining the levels of SRBC-specific antibodies remains challenging (5, 6). We have developed a simple, reliable, time- and cost-effective method to simultaneously assess levels of SRBC-specific antibodies of different isotypes in serum from immunized mice. In addition, we present an assay to stain for SRBC-binding B cells. This stain can be included in a multi-parameter circulation cytometric assay, therefore providing the possibility to study the fate and phenotype of antigen-binding B cells after SRBC immunization. In summary, these two fresh methods provide novel tools to study GC responses. Material and Methods Mice Female and male adult wildtype mice of combined FVB and C57/BL6 background were utilized for all experiments. Animals were maintained in the animal facility of the Sanford Burnham Prebys Medical Finding Institute (La Jolla, USA) or the animal facility of the Maximum Planck Institute of Immunobiology and Epigenetics (Freiburg, Germany). Protocols were authorized by the Institutional Animal Care and Use Committee and were carried out in accordance with institutional recommendations and regulations. SRBC immunization 1ml citrated CX-5461 inhibition sheep blood (Colorado Serum Organization, Denver, CO or Cedarlane, Burlington, Canada) was washed twice with 50 ml PBS and resuspended 1:10 in PBS (0.4 ml packed SRBC and 3.6 ml PBS). 100 l of the SRBC-suspension was i.p. injected into mice. Mice were sacrificed 7 or 9 days after the immunization. Blood was collected for serum on the day of immunization (d0) and on the day they were sacrificed (d7.