Induction of DTH in C57BL/6 Mice

Recommended protocol for use with:


This protocol describes induction of delayed-type hypersensitivity (DTH) in female C57BL/6 mice by immunization with mBSA emulsified in complete Freund's Adjuvant (CFA).

Note - This protocol may be used with other mouse strains, but we have tested it at Hooke only in C57BL/6 mice.

Materials needed

Qty Description
1 Hooke Kit™ mBSA/CFA Emulsion (EK-0133)
1 Methylated bovine serum albumin (mBSA) in PBS (DS-0162)
20 C57BL/6 mice, females, 9 to 14 weeks old
(Taconic Biosciences model B6-F or Jackson Laboratory strain C57BL/6J)
Phosphate buffered saline (PBS)
(standard formulation, pH 7.4, calcium-free, magnesium-free)


Mice should be acclimated at your facility for at least 7 days before immunization.

Inject mice with antigen emulsified in CFA subcutaneously at two sites in the lower back, administering 0.05 mL over each hip (total of 0.1 mL 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.

For evaluation of DTH, 5 days after immunization mice are challenged with mBSA in phosphate buffered saline (PBS, Hooke cat. no. DS-0162). Typically, mBSA in PBS is injected into footpad or ear and the counter-lateral footpad or ear (20 µL/footpad, 10 µL/ear) is injected with only PBS, as a negative control.

The thickness of paws or ears is measured with calipers, 24 hours after the challenge.

Note - Intradermal injection (as described for BALB/c mice in other published protocols) is not necessary in C57BL/6 mice. Subcutaneous injection of mBSA/CFA is technically easier than intradermal injection and produces an excellent DTH response in C57BL/6 mice.


Yoshimoto T et al, Blood 95:2869 (2000)
Särnstrand B et al, J Pharmacol Exp Ther 288:1174 (1999)
Kim YS et al, J Immunol 160 :5742 (1998)
Magram J et al, Immunity 4 :471 (1996)

Version: 2017-05