Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA122087

Local Sample ID:	S4_3
Subject ID:	SU001512
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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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).