After centrifugation at 18,630 × g for 10 min at 4°C, 0.7 ml of pk-CFS and bf-CFS samples were added to 0.1 ml of a D2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 10 mM, set to pH 7.0 by means of a 1 M phosphate buffer. The solution contained also NaN3 2 mM, to avoid microorganisms’ proliferation. 1H-NMR spectra were recorded with an AVANCE III spectrometer (Bruker, Milan, IT, United States) at 298 K operating at a frequency of 600.13 MHz. To reduce broad signals caused by slowly tumbling molecules, a CPMG filter of 400 echoes, separated by an echo time of 400 μs, was applied. Water residual signal was reduced by presaturation. The signals were assigned by comparing their chemical shift and multiplicity with Chenomx software data bank (Chenomx Inc., Canada, ver 11.05) (Laghi et al., 2014 (link)). Metabolite concentrations were calculated and reported as differences with respect to MRS medium.
Software data bank
Manufactured by Chenomx
Sourced in Canada
The Chenomx software data bank is a comprehensive collection of reference data for nuclear magnetic resonance (NMR) spectroscopy. It provides a database of metabolite information, including chemical shifts, coupling patterns, and metabolite concentrations, for a wide range of compounds commonly found in biological samples.
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Lab products found in correlation
5 protocols using software data bank
1
NMR Analysis of Microbial Metabolites
2
Fecal Metabolite Quantification by NMR
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The 1H-NMR spectra were adjusted for baseline irregularities as explained elsewhere (12 (link)). The signals were assigned by comparing their chemical shift and multiplicity with the Human Metabolome Database (13 (link)) and Chenomx software data bank (Chenomx Inc., Canada, version 8.1). Concentrations of molecules were calculated by employing the trimethylsilyl propionate (TSP) signal as an internal standard. In order to compensate for differences in dilution or solids content, all the spectra were normalized by means of probabilistic quotient normalization (14 (link)). The concentration of the molecules was expressed as millimoles per gram of fecal sample. Statistically significant differences were assessed by means of paired Wilcoxon–Mann–Whitney tests for paired samples (p < 0.05). Samples were considered as outliers, and therefore excluded, when their concentration at T0, at T6 or the T6 − T0 difference was outside 1.5 times the interquartile range (15 (link)).
3
Vaginal Metabolomic Fingerprinting Protocol
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Metabolomic analysis was performed starting from 700 μl of the cell-free supernatants of the vaginal swabs, according to Vitali et al. (2015) (link). The signals originating from large molecules were suppressed by a CPMG filter of 400 echoes, generated by 180° pulses of 24 μs separated by 400 μs (Ventrella et al., 2016 (link)). The signals were assigned by comparing their multiplicity and chemical shift with Chenomx software data bank (ver 8.1 Chenomx, Inc., Edmonton, AB, Canada).
4
Metabolomic Profiling of Lactobacillus Strains
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For each Lactobacillus strain 700 μl of cell free supernatant and 350 μl of cellular lysate were added to 160 μl of a D2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 6.25 mM set to pH 7.0 by means of a 100 mM phosphate buffer. 1H-NMR spectra were recorded at 298 K with an AVANCE III spectrometer (Bruker, Milan, Italy) operating at a frequency of 600.13 MHz, following the procedure previously described [27 (link), 35 (link)]. The signals were assigned by comparing their chemical shift and multiplicity with Chenomx software data bank (Chenomx Inc., Canada, ver 8.2), with standard (ver. 10) and HMDB (ver. 2) data banks. Differences in the extracellular/intracellular metabolome composition were firstly assessed by calculating the intra-groups Euclidean distance in a multidimensional space where each dimension represented the concentration of a molecule quantified in the cell free supernatant or cellular lysate. In a second time, differences in intracellular/extracellular metabolites were calculated by means of a one-tailed unpaired Wilcoxon test, through the homonym function implemented in R computational software (www.r-project.org ). A probability value for null hypothesis of 0.05 was accepted, corrected according to Bonferroni for multiple comparisons.
5
NMR Quantification of Lactobacilli Metabolites
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One ml of CFS obtained from lactobacilli was added to 160 μl of a D2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 6.25 mM set to pH 7.0 by means of a 100 mM phosphate buffer. 1H-NMR spectra were recorded at 298 K with an AVANCE III spectrometer (Bruker, Milan, Italy) operating at a frequency of 600.13 MHz. To avoid the presence of broad signals arising from slowly tumbling molecules, a T2 filter of 400 echoes, separated by an echo time of 400 μs, was applied. The signals were assigned by comparing their chemical shift and multiplicity with Chenomx software data bank (Chenomx Inc., Canada, ver 8.02). Literature on previous quantitative investigations conducted with the same technique reports a precision error below 2% [36 (link)].
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