2. Materials and methods
2.1. Preparation of rat peritoneal macrophages
Male Long–Evans Tokushima Otsuka (LETO)
rats, specific pathogen-free, and weighing 300–350
g were used. The experimental procedures were
approved by Gifu University, Animal Care and Use
Committee and were in accordance with Japanese
Department of Agriculture guidelines. The method
described by Handel-Fernandez and Lopez [19] is
adopted for collecting peritoneal macrophages. Briefly,
rats were sacrificed by cervical dislocation, placed
in such a manner that the abdomen is facing up and
completely wet with 70% ethanol. A transverse cut
was made in the inguinal area and the skin was
dissected to expose the abdominal wall which is then
soaked with 70% ethanol. About 20 ml of cold
DMEM (Sigma, St. Louis, MO, USA) containing
5% heat-inactivated endotoxin-free fetal bovine serum
(FBS) (Sigma) was injected. The needle was removed
and the abdomen was gently massaged. The medium
was drawn back and the peritoneal fluid was dispensed
into 50 ml polypropylene tubes. This procedure
was repeated two more times. The cells were
washed three times by centrifugation at 300g for 10
min at 4 jC and re-suspending them in DMEM
medium containing 5% FBS.