小鼠胰岛素耐量试验（ITT）

适用范围与方法

样本：mouse blood

葡萄糖测定

• streptozotocin-induced diabetes C57BL/6 mouse

  年龄

  age as reported in the cited study

  组织

  blood

• 高脂饮食代谢模型（C57BL/6背景）

  年龄

  age as reported in the cited study

  组织

  blood

• high-fat diet plus low-dose streptozotocin-induced type 2 diabetes C57BL/6J mouse

  年龄

  age as reported in the cited study

  组织

  blood

• db/db C57BLKS mouse

  年龄

  age as reported in the cited study

  组织

  blood

• db/db mouse

  年龄

  age as reported in the cited study

  组织

  blood

• high-fat diet-induced obesity and diabetes C57BL/6J mouse

  年龄

  age as reported in the cited study

  组织

  blood

• NOD mouse

  年龄

  age as reported in the cited study

  组织

  blood

• ob/ob mouse

  年龄

  age as reported in the cited study

  组织

  blood

试剂与材料

• 70%乙醇
• 血糖试纸
• 血糖试纸
• 胰岛素（insulin）
• 胰岛素（insulin）

实验流程

1. Fasting

   Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before ITT begins. Transfer mice to clean cage and wire top. Keep water bottles during the fasting period. 1. If using High Fat diet (HFD), save food to give back at the end of the ITT. 1. Mark tails with a sharpie for easier identification during ITT.

2. Insulin Solution prep

   Stock 1: 50ul of 100U/ml Humulin R into 4950ul of cold sterile 1X PBS. Invert solution 3-4 times to mix. Stock 2: 1ml of Stock 1 into 9ml of cold sterile 1X PBS. Invert solution 3-4 times to mix. Keep on ice until use

3. ITT

   Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of insulin (**1-1.2 units/kg**). Weight of mouse (g) x 10 = ul insulin stock 2 solution ITT - template spreadsheet.xlsx

4. ITT

   Prepare syringes using 1cc syringe and 26G needle. Load all syringes with calculated insulin dose and position in front of home cage.

5. ITT

   Prior to the delivery of the insulin solution, a time “zero” blood glucose level must be measured. Restrain mouse and clean tail with 70% ethanol/gauze. Extract a small amount of blood from the tail vein onto One Touch Ultra blood glucose strips used with the One Touch Ultra 2 blood glucose monitoring system.

6. ITT

   Administer the correct dose of Insulin solution using an IP (intraperitoneal) injection method. - Restrain the mouse. Then place the mouse is in dorsal recumbency with the head down allowing the viscera to move cranially. - Using the bend of the knee and the ventral abdominal midline as landmarks, the needle is inserted half way between the midline and lateral side of the animal at the natural bend of the knee. Insert the needle at a 30 to 45 degree angle into either the left or right lower abdominal quadrant. - Prior to injection, aspirate to make sure the needle has not penetrated a blood vessel, the intestines or the urinary bladder. When aspirating the syringe, you should see an air bubble in the hub of the needle, and not any form of fluid. - Inject and return mouse to cage.

7. ITT

   Begin timing from the point of successfully delivering the insulin solution.

8. ITT

   Test and record the blood glucose level (see step 5) at times 15, 30, 60, 90, and 120 minutes after the initial glucose delivery. Record each value on Insulin Tolerance Test record sheet (template found on step 2). Record condition of animal throughout and after procedure on the Insulin Tolerance Test recored sheet.

9. ITT

   Return food and environmental enrichments to cages.

质量控制与输出

禁食时间、胰岛素剂量与注射途径在研究期间保持一致；胰岛素工作液按源方案现配并冷藏；血糖仪与试纸匹配并按厂商要求校准和保存；逐动物记录体重、时间点与读数。

基线与负荷后 15、30、60、90、120 分钟血糖时间序列；可计算血糖下降百分比或曲线下面积。

研究设计

小鼠腹腔注射胰岛素后的耐量试验。禁食 4–6 小时；按 1–1.2 U/kg 给予胰岛素；负荷前及负荷后 15、30、60、90、120 分钟测定血糖。不同组别与样本量按具体研究设计确定，本方案不预设实验分组。

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