13c215n glycine

C2C12 myoblasts and myotubes were grown in DMEM supplemented with ¹³C₂¹⁵N-Glycine (30 mg/L, Cambridge Isotope Laboratories CAS211057-02-2) to label intracellular purine nucleotides. BAOECs were grown on cell culture inserts (0.4 μM pore, ThinCert, Greiner Bio-One). At the start of treatments, BAOEC cultures were inserted into C2C12 myotube–containing wells. Media samples were collected and analyzed by UPLC/MS.

Dual-labeled glycine (¹³C₂¹⁵N-glycine, 99% labeled on both C atoms and 99% labeled with ¹⁵N; Cambridge Isotope Laboratories) was added in situ in the end of September 2006 to 20 cm² sub-plots of undisturbed vegetation, providing 0.1 L of re-demineralized water with 0.027 g glycine, corresponding to 687 mg glycine·m⁻² (223 mg C·m⁻² or 0.016 mg glycine·g⁻¹ DW soil). The label was injected into the soil in 16 points of a grid fixed in a transparent plastic plate, just below the soil surface, by using a syringe connected to a dispenser on a 1 L glass bottle [54] (link), [55] . The soil naturally contained free amino acids, the total total amino acid N of the free amino acids was 25.7±4.5 g N·m⁻² in August 2005 [56] .
Two types of ¹³C label were mixed during this experiment in the undisturbed heath: 1. the highly enriched ¹³C label from glycine added to the soil in all plots and 2. the slightly depleted ¹³C label from the FACE CO₂ air in plots with elevated CO₂, which was sourced through the plant and made available for microbes in the soil.

[1-¹³C]Glucose (Sigma Aldrich), [¹³C₃, ¹⁵N]alanine, [¹³C₂, ¹⁵N]glycine and sodium [1-¹³C]pyruvate (Cambridge Isotopes) were ground by hand to obtain the RFP. The RRPs were prepared by dissolving 4-hydroxy-TEMPO-benzoate (Sigma Aldrich) in mixtures of toluene-d₆ (Armar Chemicals), THF-d₈ (Armar Chemicals), toluene and THF (Sigma Aldrich). The finely ground RFP were then impregnated with the RRP solution directly in the DNP sample holder with 3 μl of the RRP added per milligram of RFP. The impregnated mixture in the DNP sample holder was then sonicated for 10 s before insertion in the DNP polarizer that resulted in rapid freezing of the sample in liquid helium.