1. 溶液配制: (1). 溶DMEM 500ml; NE---A液(1μg/μl,1mg/ml) 母液0.1ml(5mg)+ ddH2O up to 5 ml ;过滤消毒,-20℃保存。 NE-DMEM(-6M) NE---A液(1μg/μl,1mg/ml) 20.5μl DMEM UP TO 100 ml 过滤消毒,4℃保存 (2). NE+CGRP-DMEM(-6M,100ng) NE---A液(1μg/μl,1mg/ml) 20.5μl CGRP-储存液 50 μl DMEM UP TO 100 ml 过滤消毒,4℃保存 (3). NE+IFN-DMEM(-6M,50ng) NE---A液(1μg/μl,1mg/ml) 20.5μl IFN-γ(25ng/μl) 25 μl DMEM UP TO 100 ml 过滤消毒,4℃保存 (4). 低血清DMEM培养基(上室) (5). 20%FBS-DMEM培养基(下室) 2. 准备 (1). 溶胶,4℃过夜(Thaw Matrigel at 4℃ overnight.) (2). 室温下基质胶易成凝胶,所以,步骤2和3种使用试管和枪头要在试验前-20C预冷。(Matrigel tends to form gel very quickly at room temerature, therefore, pipets and tips using in steps 2 and 3 have to be chilled at prior to experiements.) 3. 包被基底膜(冰上操作) (1). 用无血清的冷细胞培养基DMEM稀释Matrigel胶(10mg/ml to 5 mg/ml))(Dilute Matrigel (10mg/ml to 5 mg/ml) in serum free-cold cell culture media (RPMI1640, EMEM, DMEM, etc).) (2). 取100ul稀释胶加到24-well 细胞小室的上室中(Put 100 ul of the diluted matrigel into upper chamber of 24-well transwell) (3). 37℃孵育transwell至少4-5h (Incubate the transwell at 37C at least 4 to 5 h for gelling.) 4. 水化基底膜 用无血清培养基轻洗凝胶(Gently wash gelled matrigel with warmed serum free-culture media.) 5. 准备细胞悬液和小室 (1). 消化法从细胞培养瓶中获取细胞;(Harvest cells from tissue culture flasks by Trypsin/EDTA;) (2). 用培养基洗3遍(Wash the cells 3 times with culture media (RPMI1640, EMEM, DMEM etc) containing 1 % FBS.) (3). 重选细胞,5×105 cells/ml,1% FBS (Resuspend the cells in media containing 1% FBS at a density of 5×105 cells/ml.) (4). 上室加200 ul细胞悬液(Put 200 ul of the cell suspension onto the matrigel.) (5). 下室中加入600 ul细胞培养基,含有5 ug/ml fibronectin作为黏连亚族(lower chamber of the transwell is filled with 600 ul of culture media containing, as an adhesive subtrate.) 6. 孵育,37℃,20 to 24 h (Incubate at 37C for 20 to 24 h. 7. 染色和计数 (1). 棉签擦去上室上面的非侵袭细胞(Scrape off noninvaded cells on the top of the transwell with a cotton swab) (2). 移去transwells,倒置,风干(Remove transwells from 24-well plates and stained with Diff-Quick solution.) (3). 24孔板中加入500µl含0.1%结晶紫,将小室置于其中,使膜浸没在培养基中,37℃ 30min后取出,PBS清洗。 (4). 直径上取4个视野,照相,计数。 (5). 24孔板中加入500µl 33%醋酸,将小室置于其中,浸膜,振荡10min,充分溶解。取出小室,24孔板于酶标仪上570nm测OD值,间接反应细胞数。