Honeybee (L. the degranulation of mast cells and the production of pro-inflammatory cytokines in compound 48/80-treated pores and skin tissues. According to these results, BV may improve atopic dermatitis-related symptoms by inhibiting the mast cell degranulation and pro-inflammatory cytokine manifestation. L.) used in this study were taken care of in the National Academy of Agricultural Technology, Suwon, buy 256411-32-2 Korea. BV was collected by a bee venom collecting device (Chung Jin Biotech Co., Ltd., Ansan, Korea) inside a sterile manner under strict laboratory conditions. In brief, the BV collector was placed on the hive, and the bees were given enough electrical shocks to cause them to sting a glass plate from which dried bee venom was later on scraped off. The collected venom was purified by method of Han et al . Purified BV was stored in a buy 256411-32-2 refrigerator for later on use. BV used in the experiment was confirmed with size exclusion gel chromatography (AKTA Explorer, GE Healthcare, Pittsburgh, PA, USA) by dissolving in 0.02 M phosphate buffer with 0.25 M NaCl modified to pH 7.2 using a Superdex Peptide column (Amersham Biosciences, GE Healthcare, Pittsburgh, PA, USA). Mice Male BALB/c mice (6 weeks older, 20-25 g) were supplied from Orient Experimental Animal Breeding Center (Seongnam, Korea). All animals were housed in wire cages at 22 2C having a 12 h light-dark cycle, fed standard laboratory chow (Orient Bio Inc., Seongnam, Korea) and allowed water ad libitum. The animal protocols for this study were authorized by the boards of the Catholic University or college of Daegu and Daegu Catholic University or college Medical Center. Scratching behavior The scratching behavioral experiment in male BALB/c mice was performed according to the method of Sugimoto et al . One day before the experiment the rostral part of the pores and skin on the back of each mouse was clipped. On the test day, mice were put into acrylic cages (22 22 24 cm) for about 10 min for acclimatization. After that, mice were injected intraperitoneally with vehicle (n=8, saline 0.9%, 0.1 ml/10 g) or BV (n=8 for 0.01 mg/kg and n=8 for 0.1 mg/kg). One hour after treatment, the animals received a subcutaneous injection of compound 48/80 (50 g/50 l) in the dorsal region of the head using a 29 gauge needle. Immediately after the final injection the mice were returned to their observation cages, one mouse per cage. The scratching behavior was observed for 60 min by observers who have been unaware of the treatments. Vascular permeability of the skin After intradermal injection of compound 48/80 (50 g/50 l) into the rostral portion of pores and skin on the back, the injected site (1 1 cm) was defined with an indelible marker, and saline remedy of 2% Evans blue was injected intravenously into each animal. The animals were sacrificed 60 min later on and the scratching agent-injected site (1 1 cm) was immediately excised. The skin specimen was dissolved with 1 ml of 1 1 M KOH remedy by over night incubation and 4 ml of a mixture of 0.2 M phosphoric acid solution-acetone (5:13) was added. After strenuous shaking, the precipitates were filtered off and the amount of dye was measured colorimetrically Rabbit Polyclonal to ATP5G3. at 620 nm. buy 256411-32-2 Histology and mast cell degranulation After mice were sacrificed, the scratching agent-injected site (1 1 cm) was excised and fixed in neutral buffered formalin over night, inlayed in paraffin and stained with hematoxylin and eosin (H&E) and toluidine blue for histological analysis. The granulation of mast cells was examined using the method of Katayama et al.  with minor modifications. Granulation scores of 0, 1 or 2 2 were allocated as follows:.