胰岛分离.docVIP

Collagenase Preparation 1、Weigh out the collagenase（胶原酶） into a 50 ml conical and add isolation buffer to make the final concentration, as indicated in the table below. Dissolve（溶解、解散） the collagenase by vortexing（涡旋）.(12 mice + 2 extra mice = 14 mice x 5 ml/mouse = 70 ml total collagenase solution)(70 ml x 0.5 mg/ml or 0.3 mg/ml = 35 mg or 21 mg of collagenase P total) 2、Dispense（调剂，配剂） 2 ml of collagenase solution into each glass via（小瓶）l and place on ice. Load each 5 ml syringe（注射器） with 3 ml of collagenase add 30g needle and place in ice (prepare in hood). Distension（膨胀、扩张） 3、Just before dissection（剥离、解剖）, sacrifice animal by cervical（颈部） dislocation（脱节）. Spray（喷雾） the whole body with 70% of ETOH（乙醇）, making it completely wet. Make a V-insision starting at the genital area（性区）. Rotate（旋转） the mouse so the tail is facing away from you. 4、Remove the bowel（肠） to the left side of the open mouse. This will expose the pancreas and common bile duct（胆总管）. 5、Place a hemostat clamp（止血钳） on either side of the small-intestinal（小肠）, where the bile duct drains, leaving a small pocket for collagenase solution to enter the intestine. 6、Inflate（使膨胀） the pancreas through the bile duct with a 30g needle and 5 ml syringe containing 3 ml of cold collagenase solution, starting at the gall bladder（胆囊）. 7、Remove the pancreas from the body and place it in a siliconized vial containing 2 ml of collagenase solution. This step should be done QUICKLY and as cleanly as possible, minimizing collection of fat and connective tissue（结缔组织）. 8、Place the vial on ice and repeat step 3 with the next mouse. Digestion 9、Seal（封闭） each vial and place it into a 37°C water-bath. Incubate for 13-17 minutes (time varies with strain and age of the animal). 10、After time has elapsed, shake the vial vigorously（剧烈地）. The pancreas should fall apart. 11、Pour each digest through a large sterile sink strainer into a sterile（消毒） 1000 ml beaker（烧杯、量杯） and forcefully pipette（吸管） off the screen with washing b