Standard Operating Procedure

Contact Hypersensitivity induction in mice

 

Hazards:

Ketamine and Buprenorphine are controlled substances and anyone working with these needs to take the training session and be registered the SBOP (State Board of Pharmacy).  Go to this website to register for the training https://onestop2.umn.edu/training/courseDetail.jsp?course=RA0001

EXPOSURE TO ANIMALS: Personnel involved with animals may be exposed to bites, scratches, infectious agents, and allergens.

TETANOUS: a rare but potentially fatal bacterial infection is possible after any animal bite.

See also the Boynton Health Service brochures on animal bites and allergies in this manual.

CARBON DIOXIDE: toxic gas, avoid inhaling.

SHARPS: take appropriate cautions when working with needles

 

Protection:

Wear gloves and lab coat.

Wear a face mask if youÕre allergic to mice or to prevent the onset of allergies.

Have current tetanus immunization.

If biten by a mouse, immediately wash wound with soap and water. Seek medical assistance from Boynton Health Service, if necessary (625-7900).

 

 

Waste:

Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 2nd floor of Nils Hasselmo Hall.

 

 

Spill clean-up: Isolate the area. Use appropriate tools to put materials into a convenient waste container. 

 

Procedure:

 

Prepare reagents:

-DNFB (Sigma) 0.3% v/v in acetone:olive oil (4:1) [make fresh each time]

-DNFB 0.15% in acetone:olive oil (4:1)

-Ketamine xylazine mix: load 120ul/mouse into a 1cc syringe

-Buprenorphine: dilute stock 1:10 in PBS, load 30ul/mouse into a 0.3 cc syringe

-Eye moisture salve, electric shaver

 

  1. Anesthetize and shave Inject animals with 80ul ketamine/xylazine mix i.p. (use 1 cc tuberculin syringe, with 28g needle)

            Note: as this anesthetic is a controlled substance, the lab stock is kept in a locked box under JieÕs bench.  Code (now) is 1-1-2-4.  From commercial stocks of 100 mg/ml Ketmine and 100 mg/ml xylazine, technicians made stocks that are 18 mg/ml ketamine and 1.8 mg/ml xylazine (10ml ketamine + 1ml xylazine into 44ml PBS).  These are in 15 ml conical tubes.  The precise amount used should be recorded in the Disposition Log in the Controlled substances notebook above JieÕs desk.

 We are approved to inject 100ul of this i.p., which is 90 mg/kg ketamine for the average mouse (25 g).  Use 80ul to start.  If after 5 minutes, the animal is still not completely under, then a small amount more can be injected. Apply eye moisture salve to the eyes of anesthetized animals.  Shave the dorsal skin (approx. 4 cm x 2 cm).

 

  1. Prime (next day).  Immobilize awake mouse in hand. Apply 25ul of 0.3% DNFB to the shaved area with a yellow tip, moving the tip around while you dispense. Put animal back.

 

  1. Make baseline ear measurement (4 days after priming):  Anesthetize mice, as above.  Use spring-loaded micrometer (caliper) from Kaplan or Jenkins lab (whichever one you use, use the same one to measure the challenge).  Center the instrument on the right ear, and record measurement (metric). 

 

  1. Challenge (5 days after priming):  Immobilize awake mouse. (Never do this under anesthesia, as this may result in non-specific inflammation). Apply 5ul of 0.15% DNFB on the right ears of non-anesthetized animals.  Use p20 pipetteman, apply to one spot on the inside only . 

 

 

  1. Measure ear swelling (24, 48, 72 hours after challenge) Anesthetize mice, as above. Center the instrument on the right ear, and record measurement (metric).

 

Experimental design notes: Be sure to include control animals that are shaved and anesthetized but not primed or challenged (negative control), animals that are primed and challenged (positive control) and animals that are challenged but not primed (priming only control).  Use at least 3 aged matched (littermate) mice per group, preferably females.