Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA122097

Local Sample ID:	U5_2
Subject ID:	SU001512
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

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Subject:

Subject ID:	SU001512
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Mammals

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
U5_2	SA122097	FL014930	Unstimulated	Group

Collection:

Collection ID:	CO001507
Collection Summary:	Bone marrow-derived MSCs (RoosterBio Inc., Lot #000139) were expanded for two passages in culture after being received. They were frozen in ~5x105 aliquots in Cryostor CS10 freeze media (BioLife). Frozen aliquots were revived and plated in tissue culture polystyrene flasks (Corning) for 3-4 days prior to seeding onto test surfaces. MSCs were cultured in low-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, lot E16063), and 1% antibiotic/antimycotic solution (Gibco). Once confluent, MSCs were washed with sterile-filtered phosphate-buffered saline (PBS, Thermo Fisher) and detached from flasks using TrypLE express (Thermo Fisher). Dissociated cells were counted using a hemacytometer and replated at 13,000 cells/cm2 in T-75 tissue culture flasks. After overnight incubation, MSCs then were exposed to 48 hours of culture media (control conditions), or culture media supplemented with 50 ng/mL IFN- γ (Thermo Fisher). MSCs were then washed with PBS and trypsinized as described above, and the number of harvested cells counted using a hemocytometer.
Sample Type:	Mesenchymal stromal cells

Treatment:

Treatment ID:	TR001527
Treatment Summary:	MSCs were then washed with PBS and trypsinized as described above, and the number of harvested cells counted using a hemocytometer. MSCs were then resuspended in 155 mM ammonium acetate (Fluka) at a concentration of 1.6x106 cells/mL and aliquoted into 50-µL samples (8x104 cells per aliquot). Cells were then quenched by adding 200-µL MeOH into each sample vial and stored at -80 C until metabolite extraction. Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Treatment ID:

TR001527

Treatment Summary:

MSCs were then washed with PBS and trypsinized as described above, and the number of harvested cells counted using a hemocytometer. MSCs were then resuspended in 155 mM ammonium acetate (Fluka) at a concentration of 1.6x106 cells/mL and aliquoted into 50-µL samples (8x104 cells per aliquot). Cells were then quenched by adding 200-µL MeOH into each sample vial and stored at -80 C until metabolite extraction. Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Sample Preparation:

Sampleprep ID:	SP001520
Sampleprep Summary:	Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Sampleprep ID:

SP001520

Sampleprep Summary:

Frozen cells were subject to three freeze-thaw cycles, with liquid nitrogen for freezing and ice-water sonication for thawing. Cell samples were then centrifuged at 14,800 rpm for 5 min to precipitate proteins. From the supernatant, 200 µL was transferred into a new vial for lyophilization. The pooled QC sample was formed by mixing 30 µL of each sample. All cell extracts and the QC sample were then lyophilized at -40 C and 100 mTorr for 24h in a VirTis Benchtop free-drier (LP Industries, Stone Ridge, NY, USA). Residues were reconstituted in a 5.9 ×10-5M 13C-phenylalanine methanolic solution to a final volume of 10 µL (for samples), and 18 µL (for QCs).

Combined analysis:

Analysis ID	AN002402
Chromatography ID	CH001765
MS ID	MS002243
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	none
Column	none
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE
Units	Normalized Intensity (Intensity ratio against internal standard signal))

Chromatography:

Chromatography ID:	CH001765
Instrument Name:	none
Column Name:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS002243
Analysis ID:	AN002402
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	See attached Method file
Ion Mode:	POSITIVE