The metabolic alterations in media were measured with
proton NMR spectroscopy
After recovery, the culture media of SKOV3 cells were collected. One day
before the NMR experiment, media samples were thawed at the room
temperature. The sample with 540 μL was collected and mixed with 60 μL
potassium phosphate buffer (pH = 7.4) containing 1.5 M
KH2PO4, 1mM NaN3, 0.1%
TSP and D2O. A total of 580 μL mixture was placed into
an NMR tube (Bruker Corporation, Rheinstetten, Germany) with an outer
diameter of 5 mm. 1H-NMR spectra of media samples were
obtained using a Bruker 600 MHz spectrometer (Bruker Corporation,
Rheinstetten, Germany) at the operating 1H frequency
of 600.13 MHz at a temperature of 300 K. A standard NMR pulse sequence
(recycle delay-90º-t1-90º-tm-90º
acquisition) was applied to acquire 1H-NMR spectral
data (t1 = 3 μs, tm = 100 ms). The water
peak suppression was achieved using selective irradiation during a
recycle delay of 4 s and tm. A 90º pulse was adjusted to
~10 μs. A total of 32 scans for cell media were
collected into 64 k data points with a spectral width of 20 ppm.1H-NMR spectral data were acquired using TopSpin 4.0.9
(Bruker Corporation, Rheinstetten, Germany). The spectral data were
imported into MATLAB R2018a (MathWorks, Natick, Massachusetts, USA) and
SIMCA (Sartorius, Gottingen, Germany) for multivariate statistical
analysis. The chemical shift ranged from 4.7 to 5.0, and from -1 to 0.3
was cut. The spectral data was aligned with recursive segment-wise peak
alignment method and normalised with probabilistic quotient
normalisation method (Dieterle, Ross, Schlotterbeck, & Senn, 2006;
Veselkov et al., 2009). Metabolites were identified using Chenomx
software (CHENOMX, Edmonton, Canada).