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HMDPpheno1 project protocol

Bone mineral density in males of 96 strains of mice in the Hybrid Mouse Diversity Panel (HMDP)   (2011)

Farber CR, Bennett BJ, Orozco L, Eskin E, Lusis AJ
With: Zou W, Lira A, Kostem E, Kang HM, Furlotte N, Berberyan A, Ghazalpour A, Suwanwela J, Drake TA, Wang QT, Teitelbaum SL




Farber_Protocol

Project protocol — Contents
Workflow and sampling
Equipment and supplies
Procedures
Definitions and calculations
Data
References

Workflow and sampling

Workflow

Step
Accomplished procedure
Equipment
Data recorded
1
 Mice acclimated to facility
-
-
2
 Mice sacrificed and stored at -20°C
freezer
-
3
 Mice scanned
DXA
whole body, lumbar spine, and left femur BMD

Equipment and supplies

  • freezer at-20°C/refrigerator at 4°C
  • DXA scanning by PIXImus: Lunar PIXImus II Densitometer is used to assess BMD. This methodology has been validated in small animals (see Donahue1, and 2).

Reagents and solutions

cleaning and disinfecting solutions

Procedure

a. The PIXImus II is calibrated daily using a phantom of known bone density.
b. Mice are sacrificed and their carcasses stored at -20°C.
c. Prior to BMD scans, carcasses are thawed overnight at 4°C.
d. BMD scans are performed using a Lunar PIXImus II Densitometer.
e. BMD is calculated for the entire carcass minus the skull, the lumbar spine and the left femur.

Safety

For safety, gloves must be worn and radiation safety guidelines are strictly adhered to, such that technicians must be 6 feet away from the PIXImus machine during scanning.

Definitions and calculations

Bone area measurement is generated by outlining or specifying the limits or dimensions of the entire skeletal bone regions of the body (left femur, lumbar spine, and whole body), excluding the head, as regions of interest (ROI) following a full body X-ray scan.

Bone mineral content (BMC) is generated from PIXImus density scans which are assessed for accuracy using a set of 0.0 mg to 2,000 mg of hydroxyapatite standards. According to the DXA system, bone mineral content (measured as the attenuation of the X-ray by the bones being scanned) is divided by the area (also measured by the machine) of the site being scanned to obtain bone mineral density (BMD):

BMD = Bone mineral density = BMC ÷ Area [g/cm2]

Hybrid mouse diversity panel (HMDP): consists of a population of over 100 inbred mouse strains selected for usage in systematic genetic analyses of complex traits; 29 classic parental inbred and panels of recombinant inbred (RI) mice, including the BXD, CXB, BXA/AXB, and BXH panels.

Data collected by investigator

  • bone mineral density (BMD) of left femur
  • bone mineral density (BMD) of lumbar spine
  • whole body bone mineral density (BMD; skull, lumbar spine, left femur excluded)