Prepare work area in tissue culture hood by rinsing with EtOH and laying down sterile drape. Position mouse on back on sterile drape. Make a 4-5mm midline incision the skin above the sternum.
Using a 500ul syringe with a 28-gauge needle mounted in a Hamilton dispenser, inject both thymic lobes with 10ul of cells by intercostals injection between the third and fourth ribs, 3mm from the midline. Injection may be considered successful as long as the needle does not pass through the thymus. The specific depth (~2-3mm) is difficult to state due to the enormous size difference of the thymus throughout aging.
Alternatively, position the needle from craniolateral to caudomedial and inject through the thoracic wall above the clavicle. Injection may be considered successful as long as the needle does not pass through the thymus.
If necessary, close wound with surgical clips.
Ketamine/Xylazine Anesthetic Preparation:
Stock Solution: To 10ml of 100mg/ml ketamine add 2ml of 20mg/ml xylazine to make a solution of 83.3mg/ml ketamine and 3.3mg/ml xylazine. If xylazine is only available at 100mg/ml, it may be diluted 1:5 with distilled water (1ml of 100mg/ml xylazine and 4ml distilled water to give 20mg/ml xylazine.)
Working Solution: Dilute stock solution 1:3 with distilled water (4ml ketamine/xylazine solution and 8ml distilled water) to give 27.77/1.11mg/ml ketamine/xylazine working solution.
Ketamine/xylazine anesthetic should be administered at 173/7mg/kg mouse weight, or ~100ul for a 16gm mouse or ~125ul for a 20gm mouse.
References:
Goldschneider I, et al. J Exp Med. 163:1-17. Jan 1986.
Petrie HT, et al. J Exp Med. 172:1583-1588. Dec 1990.
UNIVERSITY
AND MEDICAL CENTER
