小鼠口服葡萄糖耐量试验（OGTT）

适用范围与方法

样本：mouse blood and plasma

葡萄糖耐量试验

• NOD mouse

  年龄

  age as reported in the cited study

  组织

  blood and plasma

• db/db mouse

  年龄

  age as reported in the cited study

  组织

  blood and plasma

• 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 and plasma

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

  年龄

  age as reported in the cited study

  组织

  blood and plasma

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

  年龄

  age as reported in the cited study

  组织

  blood and plasma

• streptozotocin-induced diabetes C57BL/6 mouse

  年龄

  age as reported in the cited study

  组织

  blood and plasma

试剂与材料

• D-葡萄糖（D-glucose）
• 高脂饲料 (60% kcal fat)
• 70%乙醇
• 血糖试纸
• STELLUX Chemi Rodent Insulin ELISA（ALPCO 80-INSMR-CH01）

实验流程

1. Fasting

   Fasting begins first thing in the morning (around 9am). Fast mice for 4-6 hours before OGTT 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 OGTT. 1. mark tails with a sharpie for easier identification during OGTT.

2. OGTT

   Weigh mouse to obtain body weight for dose calculations. Calculate bolus dose of glucose (**1g/kg**). Weight of mouse (g) x 2 = ul sterile glucose (50% dextrose) solution *If using HFD for 4 weeks or more, use 0.5g/kg (weight of mouse (g) x 1 = ul sterile glucose (50% dextrose) solution) GTT- template spreadsheet.xlsx

3. OGTT

   Prepare syringes using 1cc syringe and 18G oral gavage needle. Load all syringes with calculated glucose dose and position in front of home cage.

4. OGTT

   Prior to the delivery of the sterile glucose (50% dextrose) 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. Blood can be collected into microvette tubes that can be used to extract plasma which can be assayed for plasma insulin (see step 9 for plasma collection). Microvette tubes are blood collection tubes with a capillary. Tap tube until all the blood is collected in the tube. Place on ice until plasma collection.

5. OGTT

   Administer the correct dose of sterile glucose (50% dextrose) solution using the syringe and gavage needle. - Restraint of the Mouse, avoiding an excessively tight grip of the mouse, as this will induce a panic response. Ensure that the mouse cannot bite you and cannot move its head. - Gravity alone should be used to move the cannula down the esophagus. While gliding the cannula, rotate slightly as the cannula goes past the epiglottis and into the esophagus.The mouse should swallow the cannula, and this can be observed. - Inject the fluid slowly. - The cannula is then gently removed and the animal is returned to its cage.

6. OGTT

   Begin timing from the point of successfully delivering the sterile glucose (50% dextrose) solution.

7. OGTT

   Test and record the blood glucose level (see step 4) at times 15, 30, 60, 90, and 120 minutes after the initial glucose delivery. Record each value on Glucose Tolerance Test record sheet (template found on Step 2). Record condition of animal throughout and after procedure on the Glucose Tolerance Test record sheet. Extract a small amount of blood from the tail veil at 15, 30, 60 and 120 minutes as well as testing the blood glucose level. *skip blood collection at 90 minutes to ensure not to exceed 0.007 ml blood/ g body weight.

8. OGTT

   Return food and environmental enrichments to cages.

9. Plasma collection

   Place tubes into pre-chilled centrifuge. Centrifguge for 2 mins at 2000 rpm. 00:02:00 4 °C

10. Plasma collection

    Remove the callipary-lid and dispose in biohazard bin. Place the base lid and seal the microvette (click position).

11. Plasma collection

    Centrifuge at 4°C for 10 mins at 10 000 rpm. 00:10:00 4 °C

12. Plasma collection

    Using a pipette, transfer the plasma (top transparent layer) to a clean and labelled tube. Store plasma samples at -20°C until assayed.

13. Plasma collection

    Assay for insulin plasma on a STELLUX Chemi Rodent Insulin ELISA kit by ALPCO. See protocol at:

质量控制与输出

禁食时间、葡萄糖剂量与给药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；逐动物记录体重、时间点与读数；尾静脉采血总量不超过 0.007 mL/g 体重，源方案在 90 分钟时跳过采血以满足该限制；血浆离心与保存按方案条件执行。

基线与负荷后 15、30、60、90、120 分钟血糖时间序列；可计算血糖曲线下面积。

研究设计

小鼠经口灌胃葡萄糖负荷的耐受试验。禁食 4–6 小时；普通饮食按 1 g/kg、高脂饮食 4 周以上按 0.5 g/kg 给予 50% 葡萄糖溶液；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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