Procedure: Dual Energy X-Ray Absorptiometry (DXA) and X-Ray

1. DXA:
   1. Mice are anesthetized with ketamine-xylazine (i.p.) and monitored until they become unconscious.
   2. Mice are weighed and the length of the mouse is measured with a disinfected ruler (measure length starting from the nose at 0 cm to the beginning of the tail (accuracy within 0.1 cm)).
   3. The mouse is placed in the DXA analyzer (calibrated daily using phantoms from the manufacturer) and a snout scan is performed.
   4. The area of interest is optimized and a measure-scan performed (exposure dose is 300 µSv).
   5. Regions of interest are defined for data analysis (whole body excluding the head).
   6. The mouse may go directly to the X-Ray machine (below). If not, the mouse is removed (once the image has been captured) and is placed on a heated mat (37°C) in a clean cage and monitored closely until consciousness is regained.
   7. Raw data are transformed by accompanying software into bone and non-bone components, and fat and lean mass are determined.
2. X-Ray:
   1. An anesthetized (ketamine-xylazine) mouse is mounted onto an X-Ray permeable plate; the tail is curved along the left side of the body (used to enable identification of the X-Ray as dorsal or ventral).
   2. The plate is inserted into the X-Ray machine.
   3. The machine is set to a voltage of 25 kV with an integration time of 40 ms.
   4. An image is taken using the iX-Pect software.
   5. The mouse is removed and placed on a heated mat (37°C), in a clean cage and monitored closely until consciousness is fully gained.