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MB Sample ID: SA184044

Local Sample ID:	Day14-3
Subject ID:	SU002029
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

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Subject:

Subject ID:	SU002029
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Day14-3	SA184044	FL022651	14	Time after hypoxia (days)

Collection:

Collection ID:	CO002022
Collection Summary:	Wild-type male mice (C57BL/6J) were anaesthetized and tissues were harvested. Isolated mouse tissues were rinsed with cold PBS and immediately frozen in liquid nitrogen.
Sample Type:	Lung

Treatment:

Treatment ID:	TR002041
Treatment Summary:	In chronic hypoxia mice model, the male mice were housed in a hypoxic chamber (10 % O2) maintained using a hypoxic air generator (TEIJIN) and monitored with an O2 analyzer (JIKO-255), for 4, 14 or 28 day. Animals were fed a standard diet.

Sample Preparation:

Sampleprep ID:	SP002035
Sampleprep Summary:	Lipids of tissues were extracted by the method of Bligh and Dyer. The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol : isopropanol=1:1, and stored at −20°C. Fatty acid metabolites were further purified from tissues by solid-phase extraction using InertSep NH2 columns (GL Science) with deuterium-labelled internal standard (11(12)-EET-d11). Briefly, InertSep NH2 columns were precon- ditioned with 6mL of hexane and lipids extracted from tissues by the method of Bligh and Dyer were applied with 500μL of chloroform. Columns were then washed with 6mL of chloro- form/isopropanol (2/1, v/v), followed by the elution with diethyl ether/acetic acid (98/2, v/v).The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol:isopro- panol=1:1, and stored at −20°C. For the detection of fatty acid metabolites, chromatographic separation was performed on a ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm; Waters) maintained at 40°C using mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM ammonium acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v) containing 10 mM ammonium acetate) in a gradient programme (0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and 0.5 mL/min(24–32 min).

Sampleprep ID:

SP002035

Sampleprep Summary:

Lipids of tissues were extracted by the method of Bligh and Dyer. The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol : isopropanol=1:1, and stored at −20°C. Fatty acid metabolites were further purified from tissues by solid-phase extraction using InertSep NH2 columns (GL Science) with deuterium-labelled internal standard (11(12)-EET-d11). Briefly, InertSep NH2 columns were precon- ditioned with 6mL of hexane and lipids extracted from tissues by the method of Bligh and Dyer were applied with 500μL of chloroform. Columns were then washed with 6mL of chloro- form/isopropanol (2/1, v/v), followed by the elution with diethyl ether/acetic acid (98/2, v/v).The extracted solutions were dried up with centrifugal evaporator, dissolved in methanol:isopro- panol=1:1, and stored at −20°C. For the detection of fatty acid metabolites, chromatographic separation was performed on a ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm; Waters) maintained at 40°C using mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM ammonium acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v) containing 10 mM ammonium acetate) in a gradient programme (0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and 0.5 mL/min(24–32 min).

Combined analysis:

Analysis ID	AN003176
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu Nexera UC/s system (Shimadzu)
Column	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Ion trap
MS instrument name	ABI Sciex 4500 Qtrap
Ion Mode	NEGATIVE
Units	ng/mg tissue

Chromatography:

Chromatography ID:	CH002348
Instrument Name:	Shimadzu Nexera UC/s system (Shimadzu)
Column Name:	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:	40
Flow Gradient:	mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM ammonium acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v) containing 10 mM ammonium acetate) in a gradient programme (0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and 0.5 mL/min(24–32 min).
Flow Rate:	(0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and 0.5 mL/min(24–32 min)
Solvent A:	100% water; 0.1% acetic acid; 10 mM ammonium acetate
Solvent B:	80% acetonitrile/20% methanol; 10 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002954
Analysis ID:	AN003176
Instrument Name:	ABI Sciex 4500 Qtrap
Instrument Type:	Ion trap
MS Type:	ESI
MS Comments:	The instrument parameters of QTRAP4500 for negative ion mode were as follows: curtain gas, 10 psi; ionspray voltage, −4500 V; temperature, 600°C; ion source gas 1, 70 psi; ion source gas 2, 80 psi. and quantification was performed using MultiQuant™ software (SCIEX).
Ion Mode:	NEGATIVE