Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA023046

Local Sample ID:	C_8_r1
Subject ID:	SU000475
Subject Type:	Human Cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000475
Subject Type:	Human Cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
C_8_r1	SA023046	FL005608	H2O	Treatment
C_8_r1	SA023046	FL005608	Tumor	Growth_type

Collection:

Collection ID:	CO000469
Collection Summary:	An aliquot of the media following treatment was collected, and the remainder of the media was aspirated. The cells were washed with PBS twice, and quenched with 8mL ice-cold isotonic 0.9% (w/v) saline for 2 minutes. Total cellular content was then extracted with 1.7mL ice-cold acetonitrile/water (50:50, v/v) solution. Cell extracts were collected using a cell scraper and quickly transferred to MagNA Lyser Green Beads tubes (Roche, Indianapolis, USA) and stored in -80°C. Media was added to empty plates and incubated together with the cells for the duration of the experiment served as a blank.
Sample Type:	Fibroblasts

Treatment:

Treatment ID:	TR000489
Treatment Summary:	Monolayer cell cultures of desmoid tumors and normal fibroblasts from desmoid patients and an unaffected fibroblast cell line were grown in DMEM supplemented with 5% fetal bovine serum and maintained at 37°C in 5% CO2. Cells were divided when confluent and experiments were performed between the third and sixth passages. Approximately 10 x 106 cells were treated with 1.0uM Dasatinib (Selleck, Houston, USA) dissolved in DMSO, or 0.5uM FAK Inhibitor 14 (Cayman Chemicals Company, Michigan, USA) dissolved in water. Cells were incubated in fresh media containing the inhibitors, or vehicle, for 24 hours.

Treatment ID:

TR000489

Treatment Summary:

Monolayer cell cultures of desmoid tumors and normal fibroblasts from desmoid patients and an unaffected fibroblast cell line were grown in DMEM supplemented with 5% fetal bovine serum and maintained at 37°C in 5% CO2. Cells were divided when confluent and experiments were performed between the third and sixth passages. Approximately 10 x 106 cells were treated with 1.0uM Dasatinib (Selleck, Houston, USA) dissolved in DMSO, or 0.5uM FAK Inhibitor 14 (Cayman Chemicals Company, Michigan, USA) dissolved in water. Cells were incubated in fresh media containing the inhibitors, or vehicle, for 24 hours.

Sample Preparation:

Sampleprep ID:	SP000482
Sampleprep Summary:	Cells were homogenized on the MagNA Lyzer, with two 30-sec cycles at 2000 rpm, resting in a -20 °C chilling block for 1 min in between pulses, and centrifuged samples at 16,000 rcf for 4 min. The cell lysate was transferred to a new 2 mL Lo-Bind Eppendorf tubes, with the final cell count approximately 10x10^6 cells for each sample. Of the twenty cell lysate samples, six samples had sufficient volume for study samples and to be included in an analytical quality control (QC) total pool. Aliquots from these cell lysate samples were combined, divided into three total pool aliquots, and processed identically to the cell lysate study samples. All study and pool samples were lyophilized to dryness and reconstituted in a 0.2M phosphate buffer, pH 7.4, in D2O with 10% Chenomx ISTD.

Sampleprep ID:

SP000482

Sampleprep Summary:

Cells were homogenized on the MagNA Lyzer, with two 30-sec cycles at 2000 rpm, resting in a -20 °C chilling block for 1 min in between pulses, and centrifuged samples at 16,000 rcf for 4 min. The cell lysate was transferred to a new 2 mL Lo-Bind Eppendorf tubes, with the final cell count approximately 10x10^6 cells for each sample. Of the twenty cell lysate samples, six samples had sufficient volume for study samples and to be included in an analytical quality control (QC) total pool. Aliquots from these cell lysate samples were combined, divided into three total pool aliquots, and processed identically to the cell lysate study samples. All study and pool samples were lyophilized to dryness and reconstituted in a 0.2M phosphate buffer, pH 7.4, in D2O with 10% Chenomx ISTD.

Analysis:

MB Sample ID:	SA023046
Analysis ID:	AN000711
Analysis Type:	NMR
Num Factors:	13

NMR:

NMR ID:	NM000078
Analysis ID:	AN000711
Instrument Name:	Bruker Avance III
Instrument Type:	FT-NMR
NMR Experiment Type:	1D 1H
Spectrometer Frequency:	700 MHz