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MB Sample ID: SA019330
| Local Sample ID: | CP_RBC37_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 |
Select appropriate tab below to view additional metadata details:
Subject:
| 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 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| CP_RBC37_13C_0_2 | SA019330 | FL004729 | Mature erythrocyte | Cell type |
| CP_RBC37_13C_0_2 | SA019330 | FL004729 | C13 glucose | Glucose labelling |
| CP_RBC37_13C_0_2 | SA019330 | FL004729 | 20 hours | timepoint |
Collection:
| Collection ID: | CO000418 |
| Collection Summary: | Peripheral blood mononucleated cells were obtained from blood by Percoll density purification and CD34+ hemopoietic progenitor cells were isolated by magnetic bead separation according to the manufacturer's instructions (Miltenyi Biotec). CD34+ cells were cultured in a three-stage protocol based on the methods of 2d. Initially cells were cultured at 37 °C in a humid atmosphere of 5% CO2 at a density of 1 x 104 cells/mL and then maintained in the range of 2-10 × 105 cells/mL in IMDM (LifeTech) containing 5% (v/v) AB Serum (Interstate Companies Laboratories), 10 μg/mL Insulin (Sigma), 3 U/mL heparin (Pfizer), 200 μg/mL Transferrin (Prospec), 3 U/mL EPO (Eprex). During stage one (days 0-8) this was supplemented with 10 ng/mL SCF (GenScript) and 1 ng/mL IL-3 (R&D systems); during stage two (days 8-11) with 10 ng/mL SCF and additional 800 μg/mL transferrin and stage 3 (days 11-18) with 3 U/mL EPO and additional 800 μg/mL transferrin. Cultured reticulocytes (cRetics) were filtered at day 18 using a PALL WBF leukocyte filter. Isogenic control red blood cells (RBCs) were retained from donor blood, washed in IMDM and stored in saline-adenine-glucose-mannitol solution (SAG-M) at 4°C prior to use. Before analysis, cells were washed and cultured overnight in stage 3-supplemented IMDM (as outlined above). |
| Sample Type: | Cells |
| Collection Method: | See summary |
Treatment:
| Treatment ID: | TR000438 |
| Treatment Summary: | None |
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 |
Combined analysis:
| Analysis ID | AN000642 | AN000643 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | ZIC-pHILIC (Merck Sequant) | ZIC-pHILIC (Merck Sequant) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak height | peak height |
Chromatography:
| Chromatography ID: | CH000467 |
| Chromatography Summary: | Untargeted HILIC method |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | ZIC-pHILIC (Merck Sequant) |
| Column Temperature: | 25°C |
| Flow Gradient: | linear gradient -time, %B as follows: 0min- 80%, 15min- 50%, 18min- 5%, 21min- 5%, 24min- 80%, 32min- 80%. |
| Flow Rate: | 300 μL/min |
| Injection Temperature: | 4 C |
| Internal Standard: | internal standards (CHAPS, CAPS, PIPES and TRIS; all at 1 µM) |
| Sample Injection: | 10 μL |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS000574 |
| Analysis ID: | AN000642 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 320°C |
| Capillary Voltage: | +50V |
| Fragmentation Method: | None |
| Spray Voltage: | 3.5kV |
| Resolution Setting: | 70000 |
| Scan Range Moverz: | 85- 1275 m/z |
| Skimmer Voltage: | +20 V |
| Tube Lens Voltage: | +70 V |
| MS ID: | MS000575 |
| Analysis ID: | AN000643 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| Ion Mode: | NEGATIVE |
| Capillary Temperature: | 320°C |
| Capillary Voltage: | -50V |
| Fragmentation Method: | None |
| Spray Voltage: | 3.5kV |
| Resolution Setting: | 70000 |
| Scan Range Moverz: | 85- 1275 m/z |
| Skimmer Voltage: | -20 V |
| Tube Lens Voltage: | -70 V |
