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MB Sample ID: SA027951

Local Sample ID:CP_RBC36_G_0_3
Subject ID:SU000561
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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Subject:

Subject ID:SU000561
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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
CP_RBC36_G_0_3SA027951FL006928Mature erythrocyteCell type
CP_RBC36_G_0_3SA027951FL006928unlabelledGlucose labelling
CP_RBC36_G_0_3SA027951FL00692820 hourstimepoint

Collection:

Collection ID:CO000555
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:TR000575
Treatment Summary:None

Sample Preparation:

Sampleprep ID:SP000568
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 AN000818 AN000819
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:CH000585
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:MS000719
Analysis ID:AN000818
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
Capillary Temperature:300°C
Capillary Voltage:+50V
Fragmentation Method:None
Spray Voltage:4.0kV
Resolution Setting:35000
Scan Range Moverz:85- 1275 m/z
Skimmer Voltage:+20 V
Tube Lens Voltage:+70 V
  
MS ID:MS000720
Analysis ID:AN000819
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
Capillary Temperature:300°C
Capillary Voltage:-50V
Fragmentation Method:None
Spray Voltage:3.5kV
Resolution Setting:35000
Scan Range Moverz:85- 1275 m/z
Skimmer Voltage:-20 V
Tube Lens Voltage:-70 V
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