Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237004

Local Sample ID:	S14_B.cells_tfgKO_1E10
Subject ID:	SU002449
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Sample Preparation:

Sampleprep ID:	SP002455
Sampleprep Summary:	Glycerophospholipids (PC, PE, PI, PS, PG, PA) in B cells were analyzed by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 million cells were homogenized in 300 µl of Milli-Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 µl of the homogenate 400 µl of Milli-Q water, 1.875 ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4; Avanti Polar Lipids) were added. Lipids were extracted using the “One-Step Extraction” as described in Özbalci et al. 2013, Methods Mol Biol 1033:3-20. Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol.

Sampleprep ID:

SP002455

Sampleprep Summary:

Glycerophospholipids (PC, PE, PI, PS, PG, PA) in B cells were analyzed by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI-MS/MS) with direct infusion of the lipid extract (Shotgun Lipidomics): 14 to 45 million cells were homogenized in 300 µl of Milli-Q water using the Precellys 24 Homogenisator (Peqlab, Erlangen, Germany) at 6.500 rpm for 30 sec. The protein content of the homogenate was routinely determined using bicinchoninic acid. To 100 µl of the homogenate 400 µl of Milli-Q water, 1.875 ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PC 17:0-20:4, 132 pmol PE 17:0-20:4, 118 pmol PI 17:0-20:4, 131 pmol PS 17:0-20:4, 62 pmol PG 17:0/20:4, 75 pmol PA 17:0/20:4; Avanti Polar Lipids) were added. Lipids were extracted using the “One-Step Extraction” as described in Özbalci et al. 2013, Methods Mol Biol 1033:3-20. Dried lipid extracts were dissolved in 300 μl of methanol. 20 μl of the lipid extract in methanol were loaded into 96-well plates and diluted with 20 μl of 20 mM ammonium acetate in methanol.