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.
5 mm nmr tube
Manufactured by Bruker
Sourced in Germany
The 5 mm NMR tube is a laboratory equipment designed for use in nuclear magnetic resonance (NMR) spectroscopy. It is a cylindrical glass container with a diameter of 5 millimeters, used to hold samples for analysis in an NMR spectrometer.
Automatically generated - may contain errors
Lab products found in correlation
Avance 600 nmr spectrometer, by Bruker (1 mentions)
Txi probe head, by Bruker (1 mentions)
Xwin nmr software, by Bruker (1 mentions)
Matlab algorithm, by MathWorks (1 mentions)
Topspin 3, by Bruker (1 mentions)
Avance 3 600 mhz nmr spectrometer, by Bruker (1 mentions)
Cdcl3, by Merck Group (1 mentions)
Ram 2 ft raman module, by Bruker (1 mentions)
Opus 7, by Bruker (1 mentions)
Vertex 70 ftir spectrometer, by Bruker (1 mentions)
4 4 dimethyl 4 silapentane 1 sulfonic acid dss, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Topspin version 3, by Bruker (1 mentions)
600 mhz spectrometer, by Bruker (1 mentions)
Deuterium oxide, by Merck Group (1 mentions)
Nanosep centrifugal device, by Pall Corporation (1 mentions)
Topspin, by Bruker (1 mentions)
13 protocols using 5 mm nmr tube
1
NMR Analysis of Media Samples
2
Metabolite Profiling by NMR Spectroscopy
3
Metabolite Extraction from Tissue Samples
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Small molecule metabolites were extracted as described previously [17 (link)] with some modifications. Biopsy samples were resuspended in ice-cold 60% aqueous methanol and homogenized using a tissue homogenizer (Tissue Tearor Model 985370–395, Biospec Products Inc., Bartlesville, OK) set to 2-minute intervals of 10 seconds on and 5 seconds off. Tissues were transferred to glass tubes and lysed by sonication prior to addition of 1:1 aqueous choloroform and vortexing. Aqueous layers were collected by centrifugation and transferred to clean tubes prior to lyophilization for 4 hours with low heat. Lyophilized samples were resuspended in 550 μL of NMR buffer (10mM NaH2PO4/ Na2HPO4 containing 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 100% D2O, pH 7), assayed for pH, and transferred to 5mm NMR tubes (Bruker) prior to analysis.
4
NMR Sample Preparation for Metabolomics
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Dried water-soluble samples were dissolved in 700 µL of 2H2O (99.9 atom % D, Sigma Aldrich, St. Louis, Missouri, USA) and homogenized by vortexing for 1 min. Then, they were centrifuged (3000 rpm at 4 °C for 15 min) and 630 µL of each supernatant was added to 70 µL of potassium phosphate buffer (1.5 M K2HPO4, 100% (v/v) 2H2O, 2 mM NaN3, 5.8 mM deuterated trimethylsilyl propanoic acid (TMSP); pH 7.4). After stirring, a total of 600 µL from each mixture was transferred into 5 mm NMR tubes (Bruker BioSpin s.r.l) for the analysis.
The dried lipid extracts were dissolved in 700 µL of CDCl3 (99.8 atom % D, Sigma Aldrich) and homogenized by vortexing for 1 min. An aliquot of 600 µL from each sample was transferred into 5 mm NMR tubes (Bruker BioSpin s.r.l) for the analysis.
The dried lipid extracts were dissolved in 700 µL of CDCl3 (99.8 atom % D, Sigma Aldrich) and homogenized by vortexing for 1 min. An aliquot of 600 µL from each sample was transferred into 5 mm NMR tubes (Bruker BioSpin s.r.l) for the analysis.
5
FT-Raman Spectroscopy of Liquid Samples
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A RAM II FT-Raman Module (Bruker) on a VERTEX 70 FT-IR spectrometer (Bruker) with a nitrogen-cooled Ge-diode detector and a 1064 nm laser source was used to perform FT-Raman spectroscopic investigations. The liquid samples were measured in 5 mm NMR-Tubes (Bruker) with OPUS 7.0. To fit the obtained spectra, CasaXPS 2.3.16 PR 1.6 was used.
6
Urine Sample Preparation for NMR Analysis
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The second morning urine samples were collected (3 to 5 mL) and centrifuged at 3000× g for 5 min at 4 °C. The supernatant was collected from each sample (divided into three portions of 1 mL in 2 mL cryovials) and stored at −80 °C until NMR analysis, according to standard operational procedures (SOPs) [37 (link)]. NMR preparation. Each sample (1 mL) was thawed at room temperature and was centrifuged at 14.000× g for 10 min at 4 °C. For each sample, 540 µL of urine the supernatant was mixed with 60 µL of potassium phosphate buffer (1.5 M KH2PO3, 100% v/v D2O, 0.05 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 4%NaN3; pH 7.4) to a final total volume of 600 uL. After vicious vortex mixing, each sample mixture (600 µL) was transferred into a 5 mm NMR tube (Bruker BioSpin GmbH, Rheinstetten, Germany) for the analysis.
7
Daphnia magna Metabolite Extraction
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A subset of Daphna samples were extracted using buffer to compliment the in vivo NMR
assignments. For the extraction procedure, D. magna was flash-frozen in liquid nitrogen to halt enzymatic activity.20 (link) Metabolites were extracted using an aqueous
buffer.19 (link),20 (link) The D2O buffer composed of 0.2
M sodium phosphate dihydrate (NaH2PO4·2H2O, 99.3%, Fisher Scientific Company, Toronto, ON, Canada),
10 mg/L 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS; 97% purity,
Sigma Aldrich, St. Louis, MO, USA) to serve as an internal reference
standard, and 0.1% w/v sodium azide (99.5% purity, Sigma Aldrich)
added as a preservative100 (link) dissolved in
D2O (99.9% purity, Cambridge Isotope Laboratories, Andover,
MA, USA). For extraction of metabolites with the D2O buffer,
600 μL of the buffer solution was added to the homogenized daphnids,
vortexed for 30 s, and sonicated for 15 min. The mixture was then
centrifuged (Eppendorf 5804R, at 14 000 rpm for 20 min). The resulting
supernatant was transferred into a new 2 mL centrifuge tube and centrifuged
again (14 000 rpm for 20 min). The supernatant was then transferred
into a 5 mm NMR tube (Bruker BioSpin, Billerica, MA, USA) for NMR
analysis.
assignments. For the extraction procedure, D. magna was flash-frozen in liquid nitrogen to halt enzymatic activity.20 (link) Metabolites were extracted using an aqueous
buffer.19 (link),20 (link) The D2O buffer composed of 0.2
M sodium phosphate dihydrate (NaH2PO4·2H2O, 99.3%, Fisher Scientific Company, Toronto, ON, Canada),
10 mg/L 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS; 97% purity,
Sigma Aldrich, St. Louis, MO, USA) to serve as an internal reference
standard, and 0.1% w/v sodium azide (99.5% purity, Sigma Aldrich)
added as a preservative100 (link) dissolved in
D2O (99.9% purity, Cambridge Isotope Laboratories, Andover,
MA, USA). For extraction of metabolites with the D2O buffer,
600 μL of the buffer solution was added to the homogenized daphnids,
vortexed for 30 s, and sonicated for 15 min. The mixture was then
centrifuged (Eppendorf 5804R, at 14 000 rpm for 20 min). The resulting
supernatant was transferred into a new 2 mL centrifuge tube and centrifuged
again (14 000 rpm for 20 min). The supernatant was then transferred
into a 5 mm NMR tube (Bruker BioSpin, Billerica, MA, USA) for NMR
analysis.
8
NMR Sample Preparation for Biofluids
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NMR samples were prepared by thawing aliquots of the stored fluids followed by the addition of a suitable buffer. Plasma and serum samples were mixed with a buffer of pH = 7.4 (0.14 M Na2HPO4, 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 4% NaN3 in H2O) according to protocols reported elsewhere Buffer used for urine samples prepared in 100% D2O and pH = 7.4 (1.5 M KH2PO4, 0.05 mM DSS and 4% NaN3 2 mM). 300 μL of plasma and serum samples were mixed with 240 μL buffer and 60 μL D2O, while 540 μL of urine samples were mixed with 60 μL buffer (Suarez-Diez et al., 2017 (link)). The final step before NMR experiments was the transfer of 550 μL in a 5 mm NMR tube (Bruker BioSpin GmbH).
9
NMR Analysis of Saliva Metabolites
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Samples were processed for NMR using a previously published method (Cleaver et al., 2019 (link)). Briefly, centrifuged spent saliva supernatant and the sterile saliva sample were each mixed with TSP buffer (one part 2 mM sodium trimethylsilyl-[2,2,3,3-2H4]-propionate, 28.4 mg/ml Na2HPO4 and 5.28 mg/ml NaH2PO4 to two parts 50% by volume deuterium oxide, sample:buffer ratio 4:1) in a 5 mm NMR tube (Bruker, Germany). The tubes were sealed and analyzed at the Biomolecular Spectroscopy Centre, King's College London, UK on a 600 MHz spectrometer (Bruker) for 1H 1D-NMR and 1H-C13 1D- and 2D-NMR. The concentration of metabolites were collected using Chenomix NMR Suite version 8.5 (Chenomix Ltd., Canada). The 2D C13 spectra were analyzed using TopSpin version 3.6.2 (Bruker) and COLMAR (Bingol et al., 2014 (link)).
10
NMR Sample Preparation for MenA-CRM197
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To prepare NMR analytical
samples, 10 vials of lyophilized MenA-CRM197 were reconstituted
with 10 vials of liquid MenCWY singularly. All the them were pooled
in 8 mL glass vial (Wheaton) and finally dried under vacuum.
A monovalent MenA-CRM197 sample was also prepared by drying
under vacuum approximately 1 mg of conjugate as the saccharide content.
The lyophilized contents were solubilized in 0.6 mL of deuterium
oxide (99.9 atom % deuterium; Aldrich), mixed to obtain a uniform
concentration, and subsequently transferred to a 5 mm NMR tube (Bruker).
samples, 10 vials of lyophilized MenA-CRM197 were reconstituted
with 10 vials of liquid MenCWY singularly. All the them were pooled
in 8 mL glass vial (Wheaton) and finally dried under vacuum.
A monovalent MenA-CRM197 sample was also prepared by drying
under vacuum approximately 1 mg of conjugate as the saccharide content.
The lyophilized contents were solubilized in 0.6 mL of deuterium
oxide (99.9 atom % deuterium; Aldrich), mixed to obtain a uniform
concentration, and subsequently transferred to a 5 mm NMR tube (Bruker).
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