Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA205453

Local Sample ID:	B cells_DNT_GPL analysis 2b-S06_DNT6
Subject ID:	SU002225
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male and female

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Sample Preparation:

Sampleprep ID:	SP002231
Sampleprep Summary:	Glycerophospholipid analysis Glycerophospholipids (PCH, 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 x 106 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, 1.875ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PCH 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. Lipid extraction and Nano-ESI-MS/MS analysis were performed as previously described (Kumar et al., 2015). Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. The calculated glycerophospolipid amounts were normalized to the protein content of the tissue homogenate. Metabolomics of phosphorylated metabolites und carbonic acids Experimental Setup I: Splenic B cells were isolated, activated with LPS and viable cells, only GFP+ for DNT, were sorted after 3 days using flow cytometry. Perchloric acid extraction and metabolic profiling was performed as previously published measured by LCMS/MS on an QTrap 3200 (Sciex) (Hofmann et al., 2011). Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.

Sampleprep ID:

SP002231

Sampleprep Summary:

Glycerophospholipid analysis Glycerophospholipids (PCH, 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 x 106 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, 1.875ml of methanol/chloroform 2:1 (v/v) and internal standards (125 pmol PCH 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. Lipid extraction and Nano-ESI-MS/MS analysis were performed as previously described (Kumar et al., 2015). Endogenous glycerophospolipids were quantified by referring their peak areas to those of the internal standards. The calculated glycerophospolipid amounts were normalized to the protein content of the tissue homogenate. Metabolomics of phosphorylated metabolites und carbonic acids Experimental Setup I: Splenic B cells were isolated, activated with LPS and viable cells, only GFP+ for DNT, were sorted after 3 days using flow cytometry. Perchloric acid extraction and metabolic profiling was performed as previously published measured by LCMS/MS on an QTrap 3200 (Sciex) (Hofmann et al., 2011). Ref.: Kumar, V., Bouameur, J.E., Bar, J., Rice, R.H., Hornig-Do, H.T., Roop, D.R., Schwarz, N., Brodesser, 1120 S., Thiering, S., Leube, R.E., et al. (2015). A keratin scaffold regulates epidermal barrier 1121 formation, mitochondrial lipid composition, and activity. J Cell Biol 211, 1057–1075. Ref.: Hofmann, J., Bornke, F., Schmiedl, A., Kleine, T., and Sonnewald, U. (2011). Detecting functional groups of Arabidopsis mutants by metabolic profiling and evaluation of pleiotropic responses. 10Front Plant Sci 2, 82.