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MB Sample ID: SA256069
Local Sample ID: | Toxoplasma infected-5 |
Subject ID: | SU002649 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002649 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Toxoplasma infected-5 | SA256069 | FL032757 | Infected | Factor |
Collection:
Collection ID: | CO002642 |
Collection Summary: | At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally bled and sera was flash frozen and sent to the National Institute of Health (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on retention time and mass spectra from MassBank of North America, curated by the NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 and S3). The raw peak heights from each analytical platform were normalized to the average peak heights of the identified metabolites in uninfected group. The resulting data were analyzed for fold-change and multiple unpaired t-test and visualized using volcano plots to identify the differential expression of metabolites in response to T. gondii-induced cachexia. |
Sample Type: | Blood (serum) |
Treatment:
Treatment ID: | TR002661 |
Treatment Summary: | To generate cysts for infection, 8-10 week female CBA/J mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal injection. 4–8 weeks following infection, mice were euthanized by CO2 inhalation and cysts were harvest from brains homogenate passed through a 70 μm filter. Homegenate was washed 3 times in PBS, stained with dolichos biflorus agglutinin conjugated to FITC (Vector labs) at a 1:500 dilution. The number of cysts were determined by counting FITCpositive cysts at 20x magnification using an EVOS FL imaging system (Thermo Fisher). For experimental infections 10–14-week-old male C57BL/6 mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal infection resuspended in 200 Μl PBS per mouse using a 5G 5/8” tuberculin syringe. Prior to infection, mice were cross-housed on dirty, wood chip bedding for two weeks to normalize commensal microbiota and limit the effect of eating corn husk bedding on dietary metabolites. At experimental endpoints, mice were fasted for 4 hours and isoflurane anaesthetized to isolate sera via retro-orbital bleed and/or euthanized by CO2 asphyxiation to harvest tissues for weighing and histological analysis. |
Sample Preparation:
Sampleprep ID: | SP002655 |
Sampleprep Summary: | At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally bled and sera was flash frozen and sent to the National Institute of Health (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on retention time and mass spectra from MassBank of North America, curated by the NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 and S3). The raw peak heights from each analytical platform were normalized to the average peak heights of the identified metabolites in uninfected group. The resulting data were analyzed for fold-change and multiple unpaired t-test and visualized using volcano plots to identify the differential expression of metabolites in response to T. gondii-induced cachexia. |
Combined analysis:
Analysis ID | AN004197 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Waters Acquity |
Column | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | Waters Xevo TQ-S |
Ion Mode | UNSPECIFIED |
Units | pmol/mL of plasma |
Chromatography:
Chromatography ID: | CH003110 |
Chromatography Summary: | Low pH polar |
Instrument Name: | Waters Acquity |
Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
Column Temperature: | 65 |
Flow Gradient: | 30% B for 0.5min, a linear gradient to 40% B for 3min, 60% B over 3min, then 85% B for 4.5min |
Flow Rate: | 0.4mL/min |
Solvent A: | 60% water/40% acetonitrile |
Solvent B: | 90% isopropanol/10% methanol |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003944 |
Analysis ID: | AN004197 |
Instrument Name: | Waters Xevo TQ-S |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Lipid extraction and analysis for targeted sphingolipid analysis was done using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)69. Lipids were extracted from sera using an azeotrophic mix of isopropanol:water:ethyl acetate (3:1:6; v:v:v). Internal standards (10 pmol of d17 long-chain bases and C12 acylated sphingolipids) were added to samples at the onset of the extraction procedure. Extracts were separated on a Waters Iclass Acquity UPLC chromatography system. Mobile phases were (A) 60:40 water:acetonitrile and (B) 90:10 isopropanol:methanol with both mobile phases containing 5 mM ammonium formate and 0.1% formic acid. A Waters C18 CSH 2.1 mm ID × 10 cm column maintained at 65°C was used for the separation of the sphingoid bases, 1-phosphates, and acylated sphingolipids. The eluate was analyzed with an inline Waters TQ-S mass spectrometer using multiple reaction monitoring. |
Ion Mode: | UNSPECIFIED |