Palmitate (Sigma-Aldrich, St. Louis, MO, USA) was conjugated to fatty acid-free bovine serum albumin (Sigma-Aldrich) according to a previously described protocol [9 (link)]. Briefly, Palmitate was conjugated to bovine serum albumin at a 5:1 M ratio and then stored at −20 °C in a freezer purged with inert gas; isopropanol was used as a control. The stock solution was thawed for 15 min at 55 °C and then added to the cell cultures in serum-free Dulbecco’s modified Eagle’s medium. The final concentration of Palmitate in the medium was 400 μM.
Free bovine serum albumin
Manufactured by Merck Group
Sourced in United States
Free bovine serum albumin is a purified protein derived from bovine serum. It is commonly used as a laboratory reagent to stabilize proteins, support cell growth, and block non-specific binding in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Retinyl acetate, by Merck Group (2 mentions)
Orlistat, by Merck Group (2 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Merck Group (2 mentions)
Triolein, by Merck Group (2 mentions)
L α phosphatidylinositol, by Merck Group (2 mentions)
R bromoenol lactone r bel, by Cayman Chemical (2 mentions)
Palmitate, by Merck Group (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Free glycerol, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Ki16425, by Apexbio (1 mentions)
8 protocols using free bovine serum albumin
1
Palmitate Conjugation to Bovine Serum Albumin
2
Fiber Isolation from Muscle Biopsy
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The biopsies were put into a Ca2+‐free HEPES‐based solution for free dissection of fibres by fine forceps under a microscope. Samples were then transferred to 95% O2 and 5% CO2 gassed Ca2+‐free Krebs–Henseleit solution with collagenase Type 2 (Worthington, Lakewood, NJ) and essential fatty‐acid‐free bovine serum albumin (Sigma) that was shaken for 20 min at 37°C. Next, the biopsies were filtered through a 150 μm masked nylon mesh and thereafter centrifuged at 60 g at 20°C for 30 s, whereafter the supernatant was removed and the pellet resuspended in a HEPES‐based solution with a stepwise increase in Ca2+ concentration (0.1–1.8 mmol/L) with centrifugation between each step.
3
Agonist-induced Metabolic Regulation in Mice
4
Fluorescent K+ Sensor Bead Generation
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K+ sensor beads were generated by first covalently linking human IgG (Sigma) and fatty acid-free bovine serum albumin (Sigma) to 12.5 mg of carboxylated, 3 μm silica beads (Kisker Biotech) as previously described [2 (link), 68 (link)]. Beads were then washed once with coupling buffer (0.1 M sodium borate, pH 8.0), and twice with PBS. 20 mg of EDC (Sigma) and 25 μg of Asante Potassium Green 4, TMA+ salt (TEFLabs) were added to the beads in 1 ml of PBS, and the mixture incubated with agitation for 2 hours at room temperature, protected from light. APG4 beads were washed three times with PBS, before resuspension in 1 ml of coupling buffer containing 25 μg of Alexa Fluor 594-SE (Invitrogen), and incubation with agitation for an additional 1 hour at room temperature, protected from light. APG4/AF594 beads were washed twice with coupling buffer, and once with PBS, before final resuspension in 500 μl of PBS.
5
Lipid Metabolism Regulation in Hepatic Stellate Cells
6
Adipocyte Lipolysis Assay
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Mice were fasted for 4 h prior to tissue collection. Epididymal and subcutaneous fat pads were surgically removed and washed several times with phosphate-buffered saline. Tissue pieces (~20 mg) were incubated in low-glucose (1 g/L glucose) Dulbecco’s modified Eagle’s medium (Invitrogen) containing 2% fatty acid-free bovine serum albumin (Sigma) either in the presence or absence of 10 μM isoproterenol at 37 °C for 60 min. After this preincubation period, the fat pads were transferred into identical, fresh medium and incubated for a further 60 min at 37 °C. Thereafter, aliquots of the medium were removed and analyzed for free glycerol (Sigma) content using enzymatic assays, according to the manufacturer’s instructions. For protein determinations, fat pads were washed extensively with phosphate-buffered saline and then delipidated in chloroform:methanol (2:1) solution for 60 min at 37 °C. Fat pads were then lysed in 0.3 N NaOH, 0.1% SDS overnight at 55 °C. Aliquots of the protein lysates were used to determine protein content using the BCA reagent and bovine serum albumin as standard (Pierce). Lipolysis was performed in triplicate measurements for both basal and stimulated conditions from each tissue sample, and glycerol release was expressed as the mean of the three measurements for each sample.
7
Lipid Metabolism Regulation in Hepatic Stellate Cells
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Essentially fatty acid (FA)-free bovine serum albumin (BSA), ROH, RP,
retinyl acetate, triolein, L-α-phosphatidylinositol,
1,2-dioleoyl-snglycero-3-phosphocholine, and Orlistat were purchased from Sigma
Aldrich (St. Louis, MO). Atglistatin®, Lalistat2, and the HSL
inhibitor NNC 0076-0000-0079 (76-0079) were kind gifts from Dr. Rolf Breinbauer
(Institute of Organic Chemistry, University of Technology, Graz, Austria), Dr.
Paul Helquist (Department of Chemistry and Biochemistry, University of Notre
Dame, Notre Dame, IN), and Dr. Christian Fledelius (Novo Nordisk A/S, Novo
Nordisk Park, DK-2706 Måløv, Denmark), respectively.
(R)-Bromoenol lactone (R-BEL) was purchased from Cayman
Chemicals (Ann Arbor, MI). The human HSC line LX-2 was provided by Thierry
Claudel, PhD (Hans Popper Laboratory of Molecular Hepatology, Division of
Gastroenterology and Hepatology, Department of Medicine III, Medical University
of Vienna, Vienna, Austria) with the kind approval of Dr. Scott L. Friedman
(Icahn School of Medicine at Mount Sinai, New York, USA).
retinyl acetate, triolein, L-α-phosphatidylinositol,
1,2-dioleoyl-snglycero-3-phosphocholine, and Orlistat were purchased from Sigma
Aldrich (St. Louis, MO). Atglistatin®, Lalistat2, and the HSL
inhibitor NNC 0076-0000-0079 (76-0079) were kind gifts from Dr. Rolf Breinbauer
(Institute of Organic Chemistry, University of Technology, Graz, Austria), Dr.
Paul Helquist (Department of Chemistry and Biochemistry, University of Notre
Dame, Notre Dame, IN), and Dr. Christian Fledelius (Novo Nordisk A/S, Novo
Nordisk Park, DK-2706 Måløv, Denmark), respectively.
(R)-Bromoenol lactone (R-BEL) was purchased from Cayman
Chemicals (Ann Arbor, MI). The human HSC line LX-2 was provided by Thierry
Claudel, PhD (Hans Popper Laboratory of Molecular Hepatology, Division of
Gastroenterology and Hepatology, Department of Medicine III, Medical University
of Vienna, Vienna, Austria) with the kind approval of Dr. Scott L. Friedman
(Icahn School of Medicine at Mount Sinai, New York, USA).
8
Intracerebroventricular Administration of LPA Analogs
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LPA 18:1 (1-oleoyl-LPA; Tocris Bioscience, Bristol, UK) or ki16425 (ApexBio Technology, Houston, TX, USA) was dissolved in vehicle (fatty acid-free bovine serum albumin; Sigma-Aldrich, Madrid, Spain) at a concentration of 3% in saline (0.9% NaCl). The drugs were microinjected in the left lateral cerebral ventricle at a dose of 20 nM for LPA 18:1 or 400 nM for ki16425 (supporting information).
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