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MB Sample ID: SA177931

Local Sample ID:	UT2
Subject ID:	SU002000
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002006
Sampleprep Summary:	hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.

Sampleprep ID:

SP002006

Sampleprep Summary:

hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.