小鼠胰岛素耐量试验（ITT）

Scope and method

Sample：mouse blood

葡萄糖测定

• 饮食诱导肥胖（DIO）模型（文献参考）

  Age

  22 周饮食干预后

  Sex

  Mixed

  Tissue

  blood

• C57BL/6J

  Age

  12 weeks + 2 weeks（源方案典型安排）

  Tissue

  blood

• ob/ob (Lep^ob)

  Tissue

  blood

• db/db (Lepr^db)

  Tissue

  blood

Reagents and materials

• 血糖试纸
• 胰岛素（insulin）
• 70%乙醇
• PBS 磷酸盐缓冲液

Procedure

1. 禁食

   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. 胰岛素溶液配制

   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. 胰岛素负荷

   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

4. 胰岛素负荷

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

5. 胰岛素负荷

   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. 胰岛素负荷

   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. 胰岛素负荷

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

8. 胰岛素负荷

   Test and record the blood glucose level at 15, 30, 60, 90, and 120 minutes after the insulin injection. Record each value and the condition of the animal on the Insulin Tolerance Test record sheet.

9. 胰岛素负荷

   Return food and environmental enrichments to cages.

Quality control and readouts

禁食时间、胰岛素浓度与批号、剂量及注射途径在研究期间保持一致；血糖仪与试纸匹配并按厂商要求校准和保存；胰岛素溶液保持冰冷并按方案稀释；逐动物记录体重、时间点与读数。

基线与注射后 15、30、60、90、120 分钟血糖时间序列；可计算血糖下降幅度或曲线下面积。结果反映外源胰岛素负荷后的整体血糖反应，不单独等同于组织特异性胰岛素敏感性。

Study design

小鼠腹腔注射胰岛素后的血糖反应检测。禁食 4–6 小时；按 1–1.2 U/kg 给予胰岛素（源方案以 100 U/mL 原液经冷 PBS 两步稀释，注射体积按体重计算）；注射前及注射后 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。

Technical services