5 mm qxi probe

To determine the concentration of metabolites in the extracellular culture media of primary hepatocytes, media were collected from cells, derived from mice with different TTR genetic backgrounds, incubated for 9 h, and stored in −20 °C before ¹H-NMR spectroscopy quantification. Fully relaxed ¹H NMR spectra of extracellular media were obtained at 14.1 T, 25 °C, using a Bruker Avance 600 MHz spectrometer equipped with a 5 mm QXI probe with a z-gradient (Bruker Biospin, Karlsruhe, Germany) using standard methods [66 (link)]. Sodium fumarate was used as an internal reference (6.50 ppm) to quantify the following metabolites (multiplet, ppm): lactate (doublet, 1.33), alanine (doublet, 1.45), and acetate (singlet, 1.9). The relative areas of ¹H-NMR resonances were quantified using the curve-fitting routine supplied with the NUTSpro NMR spectral analysis program (Acorn, Livermore, CA, USA). The results are expressed as production in absolute values of µmol per mg total protein content of corresponding cells.

cAMP analogues were dissolved in D₂O to a final concentration of 10 mM. Spectra were recorded at 293 K or 298 K (S140) on a 750 MHz Bruker Avance NMR machine equipped with 5mm QXI probe (S1 Text). Reported chemical shifts are calibrated directly (¹H) or indirectly (³¹P, ¹³C) with respect to DSS. Assignments of S150 and S220 are validated with 2D TOCSY (mixing times of 20 and 100 ms) and [¹H;¹³C]-HSQC.