Mice from the different experiments were sacrificed by short exposure to CO2 and immediate exsanguination. Blood was collected in tubes containing lithium heparin (Microvette 500 LH, Sarstedt, Munich, Germany) centrifuged at 2000 g for 5 min at RT and stored at −80°C. The glycolytic quadriceps and gastrocnemius muscles, as well as liver samples, were quickly snap frozen in liquid nitrogen and stored at −80°C until further analysis.
Microvette 500 lh
Manufactured by Sarstedt
Sourced in Germany
The Microvette 500 LH is a laboratory collection tube designed for the collection and storage of small blood samples. It has a capacity of 500 microliters and is coated with lithium heparin, an anticoagulant that helps prevent blood clotting.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate salt, by Merck Group (1 mentions)
Penicillin g sodium salt, by Merck Group (1 mentions)
Sta 631, by Cell Biolabs (1 mentions)
Statstrip xpress, by Nova Biomedical (1 mentions)
Cobas c311 analyser, by Roche (1 mentions)
Microvette 500 k3e, by Sarstedt (1 mentions)
Comprehensive diagnostic profile rotor, by Abaxis (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Hemavet 950, by Drew Scientific (1 mentions)
Vetscan vs2, by Abaxis (1 mentions)
5 protocols using microvette 500 lh
1
Blood and Tissue Sample Collection
2
Plasma Extraction for LC-MS Analysis
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Streptomycin sulfate salt (Sigma-Aldrich, #S6501) and penicillin G sodium salt (Sigma-Aldrich, #P3032) were added to drinking water at the concentration of 2 g/L and 1500 U/mL, respectively. The drinking water was changed every 3 days during the 4-week long treatment. Blood from WT and SugctKO mice (5-week-old prior and 9-week-old post-treatment) was collected into lithium/heparin-coated vacutainers (Microvette 500 LH, Sarstedt, #20.1345.100) either by submandibular bleeding or by terminal cardiac puncture. Vacutainers were centrifuged for 5 min at 3000 rpm at 4 °C and supernatant was immediately frozen at − 80 °C. 200 µL of acetonitrile was added to 50 µL of blood plasma per sample. The mixture was vortexed vigorously for 1 min and incubated on ice for 10 min. Subsequently, the mixture was centrifuged at 14,000g for 15 min and the supernatant was transferred for LC–MS analysis. Blank and pooled quality control (QC) samples were prepared for instrument QC purposes.
3
Fasting Blood Analysis Protocol
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Blood samples were taken from the Vena facialis after 4 hours of fasting during the day cycle. Blood sugar was measured using a point‐of‐care testing device (StatStripXpress; Nova Biomedical, Waltham, MA, USA). Plasma was separated (centrifugation at 2,000g for 5 minutes at 20°C in Microvette 500 LH; Sarstedt, Germany) and analyzed for triglycerides, cholesterol, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), cholinesterase, and total protein (cobas c 311 Analyser; F. Hoffmann‐La Roche AG, Switzerland). Calibration and independent controls were used as recommended by the manufacturer. Plasma was diluted 1:5, and total bile acid content was measured using a colorimetric total bile acid assay kit (STA‐631; Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. Bile acid measurements were performed in duplicates. For sterole (except cholesterol) quantification, plasma was hydrolyzed and extracted as described.29 Subsequent differentiation and quantification of sterols were performed by gas chromatography/mass spectrometry (GC/MS).30
4
Blood Collection and Plasma Isolation
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Blood samples were collected on EDTA-coated tubes (Microvette 500 K3E, Sarstedt) or Heparin-coated tubes (Microvette 500 LH, Sarstedt), by mandibular puncture or by cardiac puncture on anaesthetized mice. Samples were centrifuged at +4°C during 10 min at 2,000 × g to collect plasma, and then plasma was stored at −20°C until analysis.
5
Liver Analysis in Mouse Model
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Liver was harvested when mice reached the indicated timepoints and fixed in 10% neutral buffered formalin (NBF) for histology, frozen in Tissue-Tek OCT compound (Sakura Finetek) in liquid nitrogen-cooled isopentane for cryosections, or snap frozen in liquid nitrogen for RNA and protein extraction. Blood was collected by cardiac puncture, kept in lithium heparin-coated Microvette 500 LH (20.1345.100; Sarstedt), and analysed using the Comprehensive Diagnostic Profile rotor (500–0038; Abaxis) on the Vetscan VS2 (Abaxis) for liver function, or using the Hemavet 950 (Drew Scientific) for blood leukocyte counting. Embryonic day 14.5 (E14.5) hepatoblasts and postnatal day 14 (P14) hepatocytes were isolated using a previously published protocol [78 ].
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