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MB Sample ID: SA185747

Local Sample ID:	B91
Subject ID:	SU002065
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	Balb/c
Age Or Age Range:	2 month-old
Gender:	Female
Animal Animal Supplier:	Animal Facility at the Instituto de Biología y Medicina Experimental (IByME) of Buenos Aires, in Argentina.
Animal Housing:	Animal Facility at the Instituto de Biología y Medicina Experimental (IByME) of Buenos Aires, in Argentina.
Animal Light Cycle:	12 h light/dark cycle
Animal Feed:	fed ad libitum
Animal Water:	fed ad libitum

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU002065
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	Balb/c
Age Or Age Range:	2 month-old
Gender:	Female
Animal Animal Supplier:	Animal Facility at the Instituto de Biología y Medicina Experimental (IByME) of Buenos Aires, in Argentina.
Animal Housing:	Animal Facility at the Instituto de Biología y Medicina Experimental (IByME) of Buenos Aires, in Argentina.
Animal Light Cycle:	12 h light/dark cycle
Animal Feed:	fed ad libitum
Animal Water:	fed ad libitum

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
B91	SA185747	FL022966	tumor	Sample type

Collection:

Collection ID:	CO002058
Collection Summary:	Tumor samples and axial mammary gland (MG) tissue from the same animal were excised and immediately frozen in liquid nitrogen. In parallel, a group of mice (n=6) without tumors were grown in identical conditions (+/- 20 mg MPA depot), to obtain healthy MG tissue for comparison
Sample Type:	Breast

Treatment:

Treatment ID:	TR002077
Treatment Summary:	Briefly, the parental tumor line, C4-HD, was induced by treatment of BALB/c female mouse with MPA depot and always transplanted with MPA to sustain growth (hormone-dependent; HD growth).

Sample Preparation:

Sampleprep ID:	SP002071
Sampleprep Summary:	Both tumor and MG tissue were ground using a pestle and mortar, while kept in liquid nitrogen. All tissue samples (average weight of 50 mg) were extracted using methanol: chloroform: water (1:1:0.75) and the polar phase was separated for analysis. In brief, ground tissue samples were transferred to an eppendorf tube, followed by the addition of 500 µL of cold 80% methanol, 400µL of cold chloroform and 200 µL of cold Mili-Q water, and vortex homogenisation for 60 s. Samples were left to rest on ice for 10 minutes and then centrifuged (8,000 rpm, 5 min, 23 ºC). Polar phases were separated, vacuum-dried and stored at -80ºC until analysis. At the time of NMR acquisition, the dried aqueous extracts were suspended in 600 µL of 100 mM sodium phosphate buffer (pH 7.4, in D2O containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) for chemical shift referencing), homogenized, and 550 µL were transferred to 5mm NMR tubes. All NMR spectra were acquired on a Bruker AVANCE III spectrometer (Rheinstetten, Germany) operating at 500.13 MHz for proton. Standard 1D 1H NMR spectra of aqueous extracts were recorded at 298 K with water presaturation, using the “noesypr1d” pulse program (Bruker library), with 2.34 s acquisition time, 2 s relaxation delay, 512 scans, 7002.801 Hz spectral width, and 32 k data points. Each free-induction decay was zero-filled to 64 k points and multiplied by a 0.3 Hz exponential function prior to Fourier transformation. After acquisition, spectra were manually phased, baseline-corrected, and chemical-shift referenced to TSP. For selected samples, two-dimensional NMR spectra, namely 1H-1H TOCSY (Total Correlation Spectroscopy) and 1H-13C HSQC (Heteronuclear Single Quantum Coherence) spectra were also recorded to support spectral assignment. Peak assignment was also based on comparison with data available on the Bruker BBIOREFCODE spectral database and the human metabolome database (HMDB), as well as on existing literature.

Sampleprep ID:

SP002071

Sampleprep Summary:

Both tumor and MG tissue were ground using a pestle and mortar, while kept in liquid nitrogen. All tissue samples (average weight of 50 mg) were extracted using methanol: chloroform: water (1:1:0.75) and the polar phase was separated for analysis. In brief, ground tissue samples were transferred to an eppendorf tube, followed by the addition of 500 µL of cold 80% methanol, 400µL of cold chloroform and 200 µL of cold Mili-Q water, and vortex homogenisation for 60 s. Samples were left to rest on ice for 10 minutes and then centrifuged (8,000 rpm, 5 min, 23 ºC). Polar phases were separated, vacuum-dried and stored at -80ºC until analysis. At the time of NMR acquisition, the dried aqueous extracts were suspended in 600 µL of 100 mM sodium phosphate buffer (pH 7.4, in D2O containing 0.25% 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) for chemical shift referencing), homogenized, and 550 µL were transferred to 5mm NMR tubes. All NMR spectra were acquired on a Bruker AVANCE III spectrometer (Rheinstetten, Germany) operating at 500.13 MHz for proton. Standard 1D 1H NMR spectra of aqueous extracts were recorded at 298 K with water presaturation, using the “noesypr1d” pulse program (Bruker library), with 2.34 s acquisition time, 2 s relaxation delay, 512 scans, 7002.801 Hz spectral width, and 32 k data points. Each free-induction decay was zero-filled to 64 k points and multiplied by a 0.3 Hz exponential function prior to Fourier transformation. After acquisition, spectra were manually phased, baseline-corrected, and chemical-shift referenced to TSP. For selected samples, two-dimensional NMR spectra, namely 1H-1H TOCSY (Total Correlation Spectroscopy) and 1H-13C HSQC (Heteronuclear Single Quantum Coherence) spectra were also recorded to support spectral assignment. Peak assignment was also based on comparison with data available on the Bruker BBIOREFCODE spectral database and the human metabolome database (HMDB), as well as on existing literature.

Analysis:

MB Sample ID:	SA185747
Analysis ID:	AN003235
Analysis Type:	NMR
Num Factors:	2
Num Metabolites:	37
Units:	ua

NMR:

NMR ID:	NM000223
Analysis ID:	AN003235
Instrument Name:	Spectrometer BRUKER AVANCE III TM HD 500 MHz
Instrument Type:	Other
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	500 MHz
NMR Solvent:	D2O
NMR Tube Size:	5mm
Shimming Method:	manual
Receiver Gain:	203
Temperature:	298º K
Number Of Scans:	512
Dummy Scans:	8