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MB Sample ID: SA023134

Local Sample ID:PBS_02
Subject ID:SU000477
Subject Type:Animal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Species Group:Mammal

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Sample Preparation:

Sampleprep ID:SP000484
Sampleprep Summary:Study Phenotypic Pools Thawed serum samples were vortexed and centrifuged at 5,000 rcf for 4 minutes. The following phenotypic pools were created by combining 10 µL from each phenotypic sample into a 2 mL LoBind eppendorf tube: 1. Pre-immune serum from RI 11-03A (n=12) which is a matched control serum that was collected before starting immunizations. The final volume of this phenotypic pool was 120 µL which was vortexed for 30 seconds. Two phenotypic pools (0wk-03A_Pool1 and 0wk-03A_Pool2) of 25 µL each were created. 2. Week 17 bleeds from RI 11-03A (n=12). The final volume of this phenotypic pool was 120 µL which was vortexed for 30 seconds. Two phenotypic pools (17wk-03A_Pool1 and 17wk-03A_Pool1) of 25 µL each were created. 3. RI 11-03B (n=8).The final volume of this phenotypic pool was 80 µL which was vortexed for 30 seconds. Two phenotypic pools (17wk-03B_Pool1 and 17wk-03B_Pool2) of 25 µL each were created. 4. and RI 11-03C (n=12). The final volume of this phenotypic pool was 120 µL which was vortexed for 30 seconds. Two phenotypic pools (17wk-03C_Pool1and 17wk-03C_Pool2) of 25 µL each were created. Whole Study Pooled QC Whole study pooled QC samples were prepared by combining 25 µL of each phenotypic pool sample created above into a 2 mL LoBind eppendorf tube. This QC pooled sample was then vortexed for 30 sec. Then, three whole study pooled QC samples of 25 µL each were aliquoted into 2 mL LoBind eppendorf tubes. Biocrates Plate Preparation: All samples (Phenotypic pools, whole study pooled QC and study samples) were vortexed for 10 minutes at 5,000 rpm. Samples were then centrifuged at 16,000 rcf for 4 minutes and at 4 °C. A Biocrates p180 kit was prepared following the AbsoluteIDQ™ p180 Kit metabolomics procedure. Briefly, an internal standard mix was added to 95 of the 96 wells. Next, zero samples, QC standards and calibration standards were added to their corresponding wells. The study samples, phenotypic and whole study pooled QC Samples (20 µL) were then added to the appropriate wells and dried for 30 minutes under nitrogen flow. The plate was derivatized using a 5% phenylisothiocyanate (PITC) solution in (1:1:1) ethanol:pyridine:water (v/v/v) and, then, incubated for 20 minutes at room temperature followed by a drying step under nitrogen flow. An extraction solvent (5 mM ammonium acetate in methanol) was added to all wells. The plate was then shaken and centrifuged. After centrifugation, 150 µL was removed and transferred to a second 96-well plate (LCMS plate). This second plate was diluted with 150 µL of HPLC grade water for a subsequent LCMS (MRM analysis) for measuring amino acids and biogenic amines. All wells in the original plate were diluted with 400 µL of flow injection analysis (FIA) Running Solvent for a FIA-MS (MRM analysis) for measuring lipids, acylcarnitines, and hexose.
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