Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Diet administration
• Procedure: Heart weight and femur length
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Administer diets	-	-
2	Weigh and euthanize mice	Scales	Body weight
3	Harvest heart and weigh	Scales	Heart weight
4	Harvest femur and measure	Digital calipers	Femur length

Equipment and supplies

• Balance
• Digital calipers

Reagents and solutions

• 10% formalin

Procedure: Diet administration

1. Two cohorts of DO mice were used: Generation 10 (G10) and G11. G10 mice were administered the control diet upon arrival at the test facility at 4 wks of age and maintained on that diet until 12-15 wks of age when they were euthanized. G11 mice were split into two groups and administered the control diet at 4 wks of age for 2 wks and then maintained on their respective experimental diets until 24-25 wks of age (18-19 wks on the experimental diet) when they were euthanized.
2. The high-fat, cholic acid (HFC) diet contained 20% fat, 1.25% cholesterol, and 0.5% cholic acid to induce atherosclerotic lesions.
3. The low-fat, high-protein (LFHP) diet contained 5% fat and 20.3% protein. This diet is not atherogenic.

Procedure: Heart weight and femur length

1. Mice are weighed then euthanized by CO₂ asphyxiation followed by cervical dislocation.
2. The heart is dissected from the chest cavity; atria and all adherent tissue are carefully excised prior to weighing the ventricles.
3. Right hind legs are removed and fixed in 10% formalin; afterward, the femur is dissected and measured two independent times to 0.01 mm using digital calipers.

Data collected by investigator

• Body weight
• Heart weight
• Heart weight adjusted to body weight
• Femur length
• Heart weight adjusted to femur length