Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA074510

Local Sample ID:	X01222018_Colo320_20V_Lipid_POS_5
Subject ID:	SU001137
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Genotype Strain:	ALDH1A1
Cell Passage Number:	5
Cell Counts:	4 million

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Sample Preparation:

Sampleprep ID:	SP001144
Sampleprep Summary:	Briefly, the cell culture medium was removed from the culture dishes, and the cells were rinsed three times with ice-cold DPBS. For quenching and metabolite extraction, 1 mL ice-cold methanol was added to the plate and the cells were immediately removed from the culture dish by scraping. The cell suspension was collected into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. After cell counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted to 4x106 cells/tube. The cells were lysed by the application of a freeze-thaw cycle (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), followed by sonication for 2 min. This process was repeated three times. The samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V Savant, Thermo Fisher Scientific). The samples were then reconstituted in isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® (Avanti Polar Lipids Inc.) was added to each sample as an internal standard.

Sampleprep ID:

SP001144

Sampleprep Summary:

Briefly, the cell culture medium was removed from the culture dishes, and the cells were rinsed three times with ice-cold DPBS. For quenching and metabolite extraction, 1 mL ice-cold methanol was added to the plate and the cells were immediately removed from the culture dish by scraping. The cell suspension was collected into 2 mL Protein Lobind Eppendorf tubes that were placed on ice. After cell counting (ViaStain AOPI Staining Solution,cridine orange/propidium iodide Nexcelom Bioscience, Lawrence, MA) using an automated cellometer (Cellometer K2, Nexcelom Bioscience, Lawrence, MA), the cell number was adjusted to 4x106 cells/tube. The cells were lysed by the application of a freeze-thaw cycle (freezing in liquid nitrogen for 5 min, thawing in warm water for 5 min), followed by sonication for 2 min. This process was repeated three times. The samples were then centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was transferred to 2 mL tubes and dried using a Speed Vac (SPD111V Savant, Thermo Fisher Scientific). The samples were then reconstituted in isopropanol:acetonitrile:water (2:1:1 %v/v) and 2.5 μL Splash® Lipidomix® (Avanti Polar Lipids Inc.) was added to each sample as an internal standard.