Immunization of Mice with Peptide Antigen
Recommended protocol for use with:
- Hooke Kit™ MOG35-55/CFA Emulsion (EK-0111)
- Hooke Kit™ PLP139-151/CFA Emulsion (EK-0122)
- Hooke Kit™ MBP1-17/CFA Emulsion (EK-0130)
Mice are immunized with peptide antigen emulsified in Complete Freund's Adjuvant (CFA) either to test their immune response to the peptide or to generate encephalitogenic cells for adoptive transfer EAE.
Most SJL mice immunized according to this protocol will develop some level of experimental autoimmune encephalomyelitis (EAE) on or after day 11 after immunization.
Other mouse strains immunized according to this protocol (not administered pertussis toxin) will usually not develop EAE. If EAE does develop, it will be milder than when pertussis toxin is used and the onset will be significantly later than in mice injected pertussis toxin.
Hooke Kit™ MOG35-55/CFA Emulsion (EK-0111),
Hooke Kit™ PLP139-151/CFA Emulsion (EK-0122), or,
Hooke Kit™ MBP1-17/CFA Emulsion (EK-0130)
|10||MOG35-55 antigen – female C57BL/6 mice
PLP139-151 antigen – female SJL mice
MBP1-17 antigen – female B10.PL, PL/J, or anti-MBP TCR transgenic mice
Mice should be 8-16 weeks old, acclimated at your facility for at least 4 days before immunization.
The mice can be injected either in the back or at the base of the tail. Mice are usually injected in the back when generating encephalitogenic cells. Injection at base of tail is preferred when evaluating immune responses in draining inguinal lymph nodes.
Back injection – generation of encephalitogenic T cells
Inject mice with the emulsion subcutaneously, at two sites on the back, injecting 0.1 mL at each site (total of 0.2 mL of emulsion per mouse). Keep the needle inserted into the subcutaneous space for 10 to 15 seconds after each injection to avoid leakage of the emulsion. Alternatively, a light pull on the syringe plunger will prevent leakage.
Encephalitogenic T cells can be generated by culturing splenocytes from the immunized mice 10 to 12 days after immunization (10 days for SJL mice).
Splenocytes need to be cultured for 3 days in the presence of antigen. In C57BL/6 mice immunomodulators (such IL-12, IL-23, anti-interferon gamma antibodies) must be added to the cultures to generate encephalitogenic cells. Some investigators add immunomodulators for other strains as well.
Base of tail injection – evaluation of immune responses
Inject mice subcutaneously in the base of tail, depositing the emulsion at the hair line. Only 0.05 mL/injection site should be administered. Two injections per mouse (one on each side of the tail base) should be given.
Immune response is usually evaluated 10 to 14 days after immunization by looking at antigen-specific proliferation and cytokine production of the draining lymph nodes and spleen cells.