MIME-Version: 1.0 Content-Location: file:///C:/B133AED4/ITT.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" GTT and fasting insulin

Insulin = Tolerance test (ITT)

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A.) FAST

Mice can be fasted for 4-= 5 hours to normalize dietary glucose/insulin levels.

Tag the cage cards with s= ticky notes indicating the start and end times of the fast.

Note: I= f you will be pooling data, it is helpful to always do your ITTs at the same time= of day and to use age-matched groups.

 

B.) ITT=

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Prepare the insulin

Prepare fresh insulin dil= utions immediately before going to the animal facility.

Insulin dilutions are mad= e from a 100unit/mL stock in sterile saline.

 

Important:

** The required insulin d= osage varies depending on the age and sex of the mice. An ideal insulin dosage sh= ould give you 40-50% of your initial (time 0) glucose reading at 60 minutes in control animals. Typically, young mice receive 0.8-1.0units/kg body weight, while older mice receive 1.0-1.2units/kg body weight, with female mice on t= he lower end and male mice on the higher end of the range. **

 

Insulin dilutions from a = 100 unit/mL stock:

Final dose:

0.8 un= its/kg

1.0 un= its/kg

1.2 un= its/kg

1st dilut= ion

(final concentration: <= /p>

0.8-1.2 units/mL)

0.08mL stock into 10mL final volume of saline

0.1mL stock into 10mL final volume of saline

0.12mL stock into 10mL final volume of saline

Final dilution<= /o:p>

(0.1 units/mL)

1:10 dilution into saline

1:10 dilution into saline

1:10 dilution into saline

 

 

Prepare the animals

  1. Label the mice using a marker on their tails to quickly tell them apart.
  2. Measure and record the weight of each mouse, and = nick tails with a razor blade.
  3. Prepare insulin syringes using diluted insulin. E= ach mouse should receive 1 unit (0.01cc) per gram of body weight from the final dilution. (For example a 50g mouse receives 50 units or 0.5cc). =

 

Begin the ITT

  1. Take a glucose reading and record it as time zero= .
  2. Inject insulin into IP cavity. (Animals should be injected approximately 30 seconds apart.)
  3. Sample blood glucose at 15, 30, 45, and 60 minutes using the glucometer. Mice should be sampled approximately 30 seconds apart so that the sample times are accurate for each animal.
  4. Return mice to cages with food.