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MB Sample ID: SA028904
Local Sample ID: | HC2_3 |
Subject ID: | SU000582 |
Subject Type: | Cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Cell Strain Details: | Suzuki, H., Moldoveanu, Z., Hall, S., et al. IgA1-secreting cell lines from patients with IgA nephropathy produce aberrantly glycosylated IgA1. J Clin Inv. 2008, 118, 629-639 |
Cell Primary Immortalized: | immortalized |
Cell Counts: | 1x10^7 cells / pellet |
Species Group: | Human |
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Sample Preparation:
Sampleprep ID: | SP000589 |
Sampleprep Summary: | A total of 24 cell pellets (4 replicates from 6 cell lines) were shipped to the NIH RTI-RCMRC on dry ice and immediately stored at -80oC after being inventoried for metabolomics analysis. An addition of 500 µL of ice-cold Cell Extraction buffer (0.005 tryptophan-d5 in 50:50 Acetonitre:Water) was added to tubes containing the cell pellet samples on ice. MagNA Lyser ceramic beads (~15-20, prewashed & dried) were added to the tubes and the MagNA Lyser was used to beat samples for two 30 sec pulses at 2,000 rpm and samples were placed on a cold block for 1 min between pulses. Samples were then centrifuged at room temperature at 16,000 rcf for 4 min. A 15 mL washed conical tube was labeled Total Pool and 120 µL of each sample was added to this conical tube. There were 24 samples; thus, the resulting Total Pool was 2880 µL. The Total Pool was vortexed and 150 µL of the Total Pool was transferred to pre-labeled 1.5 mL Lo-Bind Eppendorf tubes to make two sets of 5 Total Pool samples, and 3 Equilibrium samples. The 150 uL of the remaining supernatants of each sample was transferred to a new, pre-labeled 1.5 mL Lo-Bind Eppendorf tubes and capped with disposable rubber stoppers. Samples were placed at -80 °C for 1 h and lyophilized overnight. 100 µL of 95:5 Acetonitrile:Water was added to each tube and vortexed for 2 min at 5000 rpm, followed by centrifugation at room temperature at 16,000 rcf for 4 min. The supernatants were transferred to pre-labeled autosampler vials and 3 µL was injected into SYNAPT G2-Si. All samples were prepared and analyzed in a randomized order. |