Txi probe head

Media samples were prepared for NMR analyses based on protocols as described by Beckonert and colleagues [24 (link)]. Briefly, 300 μl media samples were mixed 1:1 with NMR phosphate buffer (50% v/v D20 (GOSS Scientific, UK, 0.01% v/v sodium 3-(trimethylsilyl) propionic acid 2,2,3,3-d4 ([TSP)], pH 7.4) and centrifuged at 12,470 × g for 5 minutes. Some 550 μl of sample was transferred into 5-mm NMR tubes (Bruker, Germany) and all samples from both studies were run on a Bruker Avance 600 NMR Spectrometer with TXI probe head (Bruker), using XWIN-NMR software (Bruker Biospin, Germany). ¹H NMR data were acquired by applying a standard one-dimensional (1D) pulse programme for 128 scans (after eight dummy scans), that included water irradiation during the recycle delay, set at 2 seconds (s). The pulse sequence was set to: recycle delay-90°-t-90°-tm-90°-ACQ, whereby 90° pulse length was set to between 16.5 μs, t (short delay) = 2 s, tm (mixing time) = 100 ms and ACQ (acquisition period) = at 2.73 s per scan. Spectral data underwent baseline correction, internal reference (TSP) peak calibration and phasing, using an in-house MATLAB algorithm (version R2012b, Mathworks Inc, USA) and Topspin 3.1 software (Bruker BioSpin, Germany). Water regions and HEPES buffer peaks were removed followed by to automatic spectral alignment and probabilistic quotient normalization [25 (link)] in MATLAB.