Freezer mill

Approximately one third of the proximal metaphyses of tibia (n=5) was dissected^{27 (link)} and soft tissues including periosteum and bone marrow were removed. The samples were pulverized to a fine powder under liquid N₂ using a Spex Freezer Mill (Spex, Inc., Metuchen, NJ, USA). Pulverized samples were washed with cold phosphate buffered saline (PBS) (pH 7.4), then with cold double distilled water several times and lyophilized. Aliquot of pulverized bone sample was demineralized with 0.5 M of EDTA for 2 weeks with several changes of EDTA, washed with cold distilled water exhaustively and lyophilized^{28 (link)}.

Human lung adenocarcinoma A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with dialyzed FBS (Life Technologies, Carlsbad, CA) and 10 mM [¹³C₆]-glucose for 24 h at 37 °C in a 5% CO₂ atmosphere. When the cells reached 70% confluence, the medium was aspirated and the cells were washed three times with ice-cold phosphate buffered saline (PBS) followed by subsequent quenching with cold acetonitrile (CH₃CN) to halt metabolic activity and addition of nanopure water (CH₃CN:H₂O at 2:1.5 (V/V)) to facilitate cell scraping and collection.
The liver was dissected from a PDX mouse fed with regular glucose or [¹³C₆]-glucose for 18 h, snap frozen and pulverized in liquid nitrogen into < 10 μm particles using a Spex freezer mill (Spex SamplePrep, Inc., Metuchen, NJ) to maximize extraction efficiency while maintaining biochemical integrity. Metabolites were subsequently extracted from liver tissue powder as described previously^{25 (link),26 (link)} using CH₃CN:H₂O:CHCl₃ (2: 1.5:1 volume ratio). The aqueous phase (polar metabolites) and organic phase (non-polar metabolites) were isolated and concentrated with either lyophilization (FTS Flexi-Dry, Stone Ridge, NY) or evaporation in a Speedvac device (Eppendorf, Hamburg, Germany), respectively.