Proliferative responses to viruses,

Proliferative responses to viruses, protein antigens, minor transplantation antigens, and the male H-Y antigen require in vivo immunization followed by in vitro stimulation. Furthermore, enhanced proliferative responses to those antigens that will generate primary in vitro responses (i.e., MHC antigens) can be obtained by in vivo priming. Multiple immunizations usually elevate in vitro responses. To immunize animals for in vitro secondary responses to soluble protein antigens or peptides, dissolve antigens and emulsify in complete Freunds adjuvant (UNIT 2.5). For strong responses by draining lymph node cells, immunize animals in a hind footpad. For strong responses by spleen cells, immunize intraperitoneally. Tail-base immunization also can be used as an efficient route of immunization; follow procedure for intradermal injection. To prime animals against cellular antigens, inject intraperitoneally with 1-5 × 107 cells that express the antigen. Immunization protocols are described in UNIT 1.6. Within 2 to 3 weeks after in vivo priming, in vitro responsiveness of primed T cells can usually be measured. This assay is often used as a preparation for subsequent in vitro cloning procedures (UNIT 3.14) and T cell hybridoma preparation (UNIT 3