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MB Sample ID: SA019340
Local Sample ID: | CP_Retics37_13C_0_2 |
Subject ID: | SU000424 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Cell Strain Details: | Reticulocytes |
Cell Primary Immortalized: | Primary |
Cell Counts: | 0.5 x 10e8 per sample |
Species Group: | Human |
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Sample Preparation:
Sampleprep ID: | SP000431 |
Sampleprep Summary: | Metabolism was quenched by immersion of cultures in an ethanol/dry ice bath to 0-4 °C. Cells were pelleted by centrifugation (10,000 rpm for 1 min) and washed in cold PBS (1 mL). Metabolites were extracted from 1 x 108 RBCs and 0.5 x 108 reticulocytes by addition of 300 µL chloroform/methanol/water (1:3:1 v/v) containing internal standards (CHAPS, CAPS, PIPES and TRIS; 1 µM) and left for 30 mins at 4 °C with periodic mixing and sonication. The number of cells used for RBCs and reticulocytes was based on the mean cell volume of each cell type, and ensured that the total cell pellet volume was equivalent for each sample, allowing direct comparison of metabolite concentrations from these samples. After mixing, cellular debris was removed by centrifugation (16,000 rpm for 10 mins) and the supernatant was kept at -80 °C prior to analysis. |
Processing Method: | Lysis with mixing and sonication at 4°C |
Processing Storage Conditions: | on ice or 4°C |
Extraction Method: | chloroform/methanol/water (1:3:1 v/v) |
Extract Storage: | -80°C |
Sample Spiking: | internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM) mixed in extraction solvent |
Cell Type: | Human stem cell derived reticulocytes and mature erythrocytes |