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MB Sample ID: SA188314

Local Sample ID:	CaOV3_Parent_1
Subject ID:	SU002091
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002097
Sampleprep Summary:	Cells were maintained at 60-80% confluence for 3 passages before being plated in 10cm dishes. Cell numbers were estimated from an additional dish with the same number of cells at seeding. Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabolic arrest was achieved through the addition of ap-proximately 2 mL of liquid nitrogen directly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice. Metabolite extraction was achieved through the addition of 1mL 100% ice cold methanol. Cells were lifted off of the plate using an ice cold cell scraper and transferred to a 2mL Eppendorf. An addi-tional 1mL of 100% ice cold methanol was added before snap freezing by immersion in liquid nitrogen for 3 minutes. This was followed by thawing on dry ice and vortexing to resuspend contents. Freeze/thaw process was repeated 5 times to ensure full extrac-tion of metabolites. Samples were centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained. The pellet was resuspended in 500uL of 100% ice cold methanol and freeze/thaw in liquid nitrogen was repeated 5 times. This sample was centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained and combined with previously retained supernatant. The samples were then dried in a SpeedVac Vacuum Concentrator (John Morris Scientific, RVC 2-18) at room tempera-ture, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in volumes of 20mM ammonium carbonate and acetonitrile to achieve identical concentration of biological material based on cell number estimate.

Sampleprep ID:

SP002097

Sampleprep Summary:

Cells were maintained at 60-80% confluence for 3 passages before being plated in 10cm dishes. Cell numbers were estimated from an additional dish with the same number of cells at seeding. Media was aspirated and cells were washed three times with 3mL warmed PBS. Metabolic arrest was achieved through the addition of ap-proximately 2 mL of liquid nitrogen directly to cells ensuring that the surface of the plate was covered before plates were placed onto dry ice. Metabolite extraction was achieved through the addition of 1mL 100% ice cold methanol. Cells were lifted off of the plate using an ice cold cell scraper and transferred to a 2mL Eppendorf. An addi-tional 1mL of 100% ice cold methanol was added before snap freezing by immersion in liquid nitrogen for 3 minutes. This was followed by thawing on dry ice and vortexing to resuspend contents. Freeze/thaw process was repeated 5 times to ensure full extrac-tion of metabolites. Samples were centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained. The pellet was resuspended in 500uL of 100% ice cold methanol and freeze/thaw in liquid nitrogen was repeated 5 times. This sample was centrifuged at 16000g at -9⁰C for 5 minutes and the supernatant was retained and combined with previously retained supernatant. The samples were then dried in a SpeedVac Vacuum Concentrator (John Morris Scientific, RVC 2-18) at room tempera-ture, with vacuum of 40mbar and rotor speed of 1000min-1. Before data acquisition samples were resuspended in volumes of 20mM ammonium carbonate and acetonitrile to achieve identical concentration of biological material based on cell number estimate.