Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA078849

Local Sample ID:	LOUNH91_LUNG
Subject ID:	SU001206
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001206
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
LOUNH91_LUNG	SA078849	FL012064	lung_NSC	Classifications
LOUNH91_LUNG	SA078849	FL012064	RPMI 1640 + 20% FBS	Cell Culture Media

Collection:

Collection ID:	CO001200
Collection Summary:	Cancer Cell Line Encyclopedia: a collection of 928 cancer cell lines
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001221
Treatment Summary:	No treatment applied to cells

Sample Preparation:

Sampleprep ID:	SP001214
Sampleprep Summary:	Polar metabolite extraction LC-MS grade solvents were used for all of the metabolite extraction in this study. For adherent cells, the media were aspirated off as much as possible and the cells were washed with 4 mL cold Phosphate Buffered Saline (PBS, no Mg2+/Ca2+). After vacuum aspiration of PBS, the metabolites were extracted by adding 4 mL 80% methanol (-80°C) immediately and the samples were transferred to a -80°C freezer. The flasks were kept on dry ice during the transfer and were incubated at -80°C for 15 min. Then the lysate was collected by a cell scraper and transferred to a 15 mL conical tube on dry ice. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL conical tube on dry ice and the tube with the pellet was kept for further extraction. Then, 500 μL 80% methanol (-80°C) was added to each pellet. The mixture was resuspended by vortexing or pipetting and transferred to a 1.5 ml centrifuge tube on dry ice. The cell debris was removed by centrifuging samples at 10,000 rpm for 5 min (4°C). The supernatant was transferred to the corresponding 15 mL conical tube on dry ice so that all extracts were combined. The pooled extracts were stored at - 80°C before LC-MS analysis. For cells growing in suspension, they were centrifuged to pellet at 300g for 5 min (4°C) and the supernatant was then aspirated off as much as possible. These cells were washed once with 4 mL cold PBS (no Mg2+/Ca2+) and they were pelleted at 300g for 5 min (4°C). After vacuum aspiration of PBS, the metabolites were extracted by adding 4 mL 80% methanol (-80°C) immediately and the samples were transferred to a -80°C freezer after brief vortexing. The samples were kept on dry ice during the transfer and were incubated at -80°C for 15 min. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The subsequent steps were the same as those used for adherent cell lines Lipid extraction For adherent cells, the medium was aspirated off as much as possible and the cells were washed with 4 mL cold PBS (no Mg2+/Ca2+). After vacuum aspiration of PBS, the lipid metabolites were extracted by adding 4 mL isopropanol (4°C) and the lysate was collected by a cell scraper and transferred to a 15 mL conical tube on ice. The samples were covered to avoid exposure to light and were allowed to sit for 1h at 4°C. Samples were then vortexed and the cell debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL centrifuge tube on ice and stored at -20°C before LC-MS analysis. For cells growing in suspension, they were centrifuged to pellet at 300g for 5 min (4°C) and the supernatant was then aspirated off as much as possible. These cells were washed once with 4 mL cold PBS (no Mg2+/Ca2+) and they were pelleted at 300g for 5 min (4°C). After vacuum aspiration of PBS, the lipid metabolites were extracted by adding 4 mL isopropanol (4°C) immediately. After brief vortexing, the samples were covered to avoid exposure to light and were allowed to sit for 1h at 4°C. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL centrifuge tube on ice and stored at -20°C before LC-MS analysis.

Sampleprep ID:

SP001214

Sampleprep Summary:

Polar metabolite extraction LC-MS grade solvents were used for all of the metabolite extraction in this study. For adherent cells, the media were aspirated off as much as possible and the cells were washed with 4 mL cold Phosphate Buffered Saline (PBS, no Mg2+/Ca2+). After vacuum aspiration of PBS, the metabolites were extracted by adding 4 mL 80% methanol (-80°C) immediately and the samples were transferred to a -80°C freezer. The flasks were kept on dry ice during the transfer and were incubated at -80°C for 15 min. Then the lysate was collected by a cell scraper and transferred to a 15 mL conical tube on dry ice. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL conical tube on dry ice and the tube with the pellet was kept for further extraction. Then, 500 μL 80% methanol (-80°C) was added to each pellet. The mixture was resuspended by vortexing or pipetting and transferred to a 1.5 ml centrifuge tube on dry ice. The cell debris was removed by centrifuging samples at 10,000 rpm for 5 min (4°C). The supernatant was transferred to the corresponding 15 mL conical tube on dry ice so that all extracts were combined. The pooled extracts were stored at - 80°C before LC-MS analysis. For cells growing in suspension, they were centrifuged to pellet at 300g for 5 min (4°C) and the supernatant was then aspirated off as much as possible. These cells were washed once with 4 mL cold PBS (no Mg2+/Ca2+) and they were pelleted at 300g for 5 min (4°C). After vacuum aspiration of PBS, the metabolites were extracted by adding 4 mL 80% methanol (-80°C) immediately and the samples were transferred to a -80°C freezer after brief vortexing. The samples were kept on dry ice during the transfer and were incubated at -80°C for 15 min. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The subsequent steps were the same as those used for adherent cell lines Lipid extraction For adherent cells, the medium was aspirated off as much as possible and the cells were washed with 4 mL cold PBS (no Mg2+/Ca2+). After vacuum aspiration of PBS, the lipid metabolites were extracted by adding 4 mL isopropanol (4°C) and the lysate was collected by a cell scraper and transferred to a 15 mL conical tube on ice. The samples were covered to avoid exposure to light and were allowed to sit for 1h at 4°C. Samples were then vortexed and the cell debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL centrifuge tube on ice and stored at -20°C before LC-MS analysis. For cells growing in suspension, they were centrifuged to pellet at 300g for 5 min (4°C) and the supernatant was then aspirated off as much as possible. These cells were washed once with 4 mL cold PBS (no Mg2+/Ca2+) and they were pelleted at 300g for 5 min (4°C). After vacuum aspiration of PBS, the lipid metabolites were extracted by adding 4 mL isopropanol (4°C) immediately. After brief vortexing, the samples were covered to avoid exposure to light and were allowed to sit for 1h at 4°C. The insoluble debris was removed by centrifuging at 3500 rpm for 10 min (4°C). The supernatant was transferred to a new 15 mL centrifuge tube on ice and stored at -20°C before LC-MS analysis.

Combined analysis:

Analysis ID	AN001875	AN001876	AN001877
Analysis type	MS	MS	MS
Chromatography type	HILIC	HILIC	Reversed phase
Chromatography system	Agilent 1100	Waters Acquity	Agilent 1200
Column	Unspecified	Phenomenex Luna NH2 (150 x 2.1mm,3um)	Unspecified
MS Type	ESI	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 4000 QTrap	ABI Sciex 5500 QTrap	ABI Sciex 4000 QTrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE
Units	Abundances	Abundances	Abundances

Chromatography:

Chromatography ID:	CH001356
Chromatography Summary:	HILIC-pos
Instrument Name:	Agilent 1100
Column Name:	Unspecified
Chromatography Type:	HILIC

Chromatography ID:	CH001357
Chromatography Summary:	HILIC-neg
Instrument Name:	Waters Acquity
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm,3um)
Chromatography Type:	HILIC

Chromatography ID:	CH001358
Chromatography Summary:	C4-pos
Instrument Name:	Agilent 1200
Column Name:	Unspecified
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001731
Analysis ID:	AN001875
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	n/a
Ion Mode:	POSITIVE

MS ID:	MS001732
Analysis ID:	AN001876
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	n/a
Ion Mode:	NEGATIVE

MS ID:	MS001733
Analysis ID:	AN001877
Instrument Name:	ABI Sciex 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Full scan data were acquired using Q1 scanning
Ion Mode:	POSITIVE