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MB Sample ID: SA273977

Local Sample ID:56
Subject ID:SU002832
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

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Subject:

Subject ID:SU002832
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
56SA273977FL035528Beta ThalFactor

Collection:

Collection ID:CO002825
Collection Summary:The spleen organ tissue were harvested, finely minced, and each organ placed individually in 2mL Eppendorf tubes containing 1mL of DMEM with Collagenase D (Sigma Aldrich, product #11088866001). The tissues were incubated at 37C with agitation for 30 minutes. After incubation, 100 uL of 0.1M EDTA was added to the tissue-containing tubes and placed on ice. A single cell suspension was created by addition of Hanks buffered salt solution (HBSS, Corning, product #MT21022CV), passing through a 100 um filter, and collected in a 15 mL conical tube. The cell suspension was spun at 500g for 5 minutes and the supernatant discarded. The remaining tissue/cell solution was resuspended in 5ml of RBC lysis buffer (Invitrogen eBiosciences, product #00-4333-57), incubated at room temperature for 15 minutes, and centrifuged at 500g for 5 minutes, this step was repeated if RBC presence was sustained. Next, the cells were washed with Miltenyi buffer (HBSS, 0.5M EDTA, Fetal bovine serum), resuspended in 180uL Miltenyi buffer, and incubated on ice with CD11b Microbeads (Miltenyi Biotech, product #130-093-636) for 15minutes. Positive cells were isolated using magnetic LS columns (Miltenyi Biotech, product #130-042-401), collected in 2 mL Eppendorf tubes, counted, and then final pellet aliquots were frozen in liquid nitrogen and stored at -80C. continuously using LabScribe2 and analyzed offline.
Sample Type:Macrophages

Treatment:

Treatment ID:TR002841
Treatment Summary:Eight- to ten-week-old female C57Bl/6 WT, Berk-ss, or Hbbth3/+ mice were either obtained from Jackson Laboratories (Bar Harbor, ME, USA) or our in-house Berk SCD mouse colony. Mice were housed and bred in an AAALAC accredited animal facility at the University of Colorado, Denver, Anschutz Medical campus and were maintained on a 12:12 light-dark cycle with food and water available ad libitum. Female heterozygous Berk-ss mice were bred with male homozygous Berkss mice to generate homozygous offspring. Specifically, Berk-ss mice with genotype Tg(HuminiLCR α1 Gγ Aγ δ βs ) Hba0/0 Hbb0/0 and the hemizygous with genotype Tg(Hu-miniLCR α1 Gγ Aγ δ βs ) Hba0/0 Hbb0 Hbb+ were littermates. Genotyping of mice used for breeding and experiments was performed by TransnetYX (Cordova, TN, USA). A total of 21 mice (C57Bl/6: n=10, Berk-ss mice: n=11, Hbbth3/+ =10) were used in the present investigation and levels of discomfort and distress were monitored daily by the in-house animal care staff, with a veterinarian available as needed. All experimental procedures were conducted under the guidelines recommended by The Journal of Physiology (11), the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at the University of Colorado, Denver, Anschutz Medical Campus.

Sample Preparation:

Sampleprep ID:SP002838
Sampleprep Summary:Spleen macrophages were extracted in methanol:acetonitrile:water (5:3:2 v/v/v – at a 1x10^6 cells/ml) ). The samples were vortexed for 30mins at 4°C. The samples were then spun down at 18,213 g for 10 minutes, 4°C and 50uL of supernatant was transferred to autosampler vial.

Combined analysis:

Analysis ID AN004418 AN004419
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE POSITIVE
Units Peak Area Peak Area

Chromatography:

Chromatography ID:CH003317
Chromatography Summary:Negative ion Mode
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min 100-0% B, 3-5 min hold at 0% B
Flow Rate:0.450ml/min
Solvent A:95% water/5% acetonitrile; 1 mM ammonium acetate
Solvent B:95% acetonitrile/5% water; 1 mM ammonium acetate
Chromatography Type:Reversed phase
  
Chromatography ID:CH003318
Chromatography Summary:Positive Ion Mode
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B.
Flow Rate:0.450ml/min
Solvent A:10% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS004165
Analysis ID:AN004418
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:NEGATIVE
  
MS ID:MS004166
Analysis ID:AN004419
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database.
Ion Mode:POSITIVE
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