Isolation of rat peritoneal mast cells.

Adult male SPF Wistar rats weighing 250–300 g were purchased from Clea Japan (Tokyo, Japan), maintained in a controlled environment at an ambient temperature of 22°C and a 12:12-h light-dark cycle, and deprived of food but allowed free access to water overnight before the experiment. All rats were handled according to the guidelines for the ethical use of animals as set by the U.S. National Institutes of Health, and all experiments were approved by the institutional Animal Care and Use Committee of the Graduate School of Veterinary Medicine, Hokkaido University. To isolate mast cells, rats were anesthetized by CO₂ inhalation and euthanized by exsanguination. Mast cells were obtained by peritoneal lavage after a 2-min massage of the abdomen with an intraperitoneal injection of 10 ml ice-cold phosphate-buffered saline (PBS) (15). The abdominal cavity was opened, peritoneal exudates were harvested into 15-ml Falcon tubes, and they were centrifuged at 1,400 g for 5 min after which the cell pellets were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Mast cells were morphologically identified under a light microscope by their large size and dense content of granules, whereas others, including leucocytes, were significantly smaller, and erythrocytes did not emit fluo-4 fluorescence for lack of esterase.