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MB Sample ID: SA097509
| Local Sample ID: | F1_9A1 |
| Subject ID: | SU001410 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
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Sample Preparation:
| Sampleprep ID: | SP001418 |
| Sampleprep Summary: | Mouse serum (30 µL) was added to 215 µL of ice-cold methanol containing 50 µg/mL butylated hydroxytoluene (BHT). Samples were homogenized by three rounds of vortex mixing for 30 seconds, freezing in liquid nitrogen for one minute, thawing for two minutes and sonicating for 10 minutes at 15°C, power 100% in a Grant XUB18 bath sonicator. Tissue wet weight was determined using a Mettler-Toledo XS105 balance (Mettler-Toledo, Melbourne, Australia). Biopsies were transferred to Eppendorf tubes containing 500 µL ice-cold methanol, 50 µg/ml BHT and one steel bead and homogenized in a TissueLyzer LT (Qiagen, Melbourne, Australia) for six minutes at 50 Hz. Homogenate was transferred to new tubes and the original tube was washed with 400 µL methanol and transferred. Samples were dried down under nitrogen flow and resuspended in 20 µL water and 200 µL methanol (50 µg/ml BHT). Samples were homogenized by three rounds of vortex mixing for 30 seconds, freezing in liquid nitrogen for one minute, thawing for two minutes and sonicating for 10 minutes at 15°C, power 100% in a Grant XUB18 bath sonicator. SPLASH LipidoMix Mass Spec Standard (10 µL) and Cer/Sph mixture II (10 µL) internal standards mixes from Avanti Polar Lipids were then added to each sample. After overnight incubation at -30°C, 750 µL MTBE was added and each tube was vortex mixed for 10 seconds and shaken for 10 minutes on a tube rotator (4°C). MilliQ water (188 µL) was then added, and the tube was vortex mixed for 30 seconds to form a biphasic separation. After centrifuging for 15 minutes at 15,000 × g, 700 µL of the clear upper phase containing lipids in MTBE was transferred to another tube and dried down using a gentle stream of nitrogen. After drying down of lipid extracts were resuspended in 50 µL methanol (containing 50 µg/mL BHT)/toluene (90/10%, v/v). |
| Processing Storage Conditions: | On ice |
