Primary brown preadipocytes (40,000 cells per well) were plated in a 48-well dish. 24 hours after plating brown preadipocytes were differentiated with adipogenic cocktail (1 μM rosiglitazone, 0.5 mM IBMX, 5 μM dexamethasone, 0.114 μg ml−1 insulin, 1 nM T3, and 125 μM Indomethacin). Cells were re-fed every 48 hours with maintenance cocktail (1 μM rosiglitazone and 0.5 μg ml−1 insulin). Adipocytes were fully differentiated 6 days after initial exposure to adipogenic cocktail. On day 6 post-differentiation, maintenance cocktail was replaced with 300 μl of DMEM/F-12 (ThermoFisher Scientific) supplemented with 2% fatty acid-free BSA, and different doses of NE for 1 hour. Adipocytes were washed twice with warm D-PBS and lysed with lysis solution (0.3 N NaOH, 0.1% SDS) for three hours at room temperature. Glycerol content of the samples was determined using Free glycerol reagent (Sigma) and Glycerol Standard Solution (Sigma) at an absorbance of 540 after incubating sample and standards with Free glycerol reagent for 15 minutes at room temperature in the dark.
Free glycerol reagent
Manufactured by Merck Group
Sourced in United States, Germany, Denmark, Japan, Spain, Holy See (Vatican City State)
The Free Glycerol Reagent is a laboratory product designed to measure the concentration of free glycerol in a sample. It provides a reliable and accurate method for determining the level of free glycerol, which is a useful metric in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Triglyceride reagent, by Merck Group (47 mentions)
Isoproterenol, by Merck Group (15 mentions)
Glycerol standard solution, by Merck Group (11 mentions)
Nefa kit, by Fujifilm (11 mentions)
Amyloglucosidase, by Merck Group (9 mentions)
Cl316 243, by Merck Group (9 mentions)
Tag reagent, by Merck Group (8 mentions)
Glycerol standard, by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Fatty acid free bsa, by Merck Group (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Oxidase peroxidase assay, by Merck Group (5 mentions)
Ensight multimode plate reader, by PerkinElmer (5 mentions)
Tissuelyser lt, by Qiagen (5 mentions)
Bradford assay, by Bio-Rad (5 mentions)
Forskolin, by Merck Group (5 mentions)
Nefa hr 2 kit, by Fujifilm (5 mentions)
Tween 20, by Merck Group (4 mentions)
Insulin, by Merck Group (4 mentions)
Nefa c kit, by Fujifilm (4 mentions)
282 protocols using free glycerol reagent
1
Brown Adipocyte Lipolysis Assay
2
Triglyceride Assay for Drosophila Metabolomics
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The TAG assay was adapted from [72 (link)]. Multiple batches of 10 male flies each were processed for TAG analysis. A small scoop of zirconium beads and 300 μl of PBS, 0.05% Tween was added to each tube. The samples were then homogenised using a Precellys homogeniser. For protein estimation, 40 μl of homogenised sample was immediately collected and frozen. The remaining sample was heat inactivated for 10 minutes at 70°C. A volume of 200 μl of the heat-inactivated sample was transferred to fresh tubes. A volume of 4 μl of lipase (25 KU/mL; Merck; 437707) was added to each tube and mixed. The samples were then kept at 37°C overnight. After overnight incubation, the samples were centrifuged at 14,000 rpm for 3 minutes, and the supernatant collected. Each sample was then put in triplicates in a 96-well plate. Different concentrations of glycerol were used as standards. Prewarmed Free Glycerol reagent (Merck; F6428) was then added to the 96-well plate, and the plate was incubated at 37°C for 6 minutes. After a brief spin, absorbance measurements were taken at 540 nm using a Hidex Sense Plate Reader. The Bradford assay was used for protein estimation. Briefly, pre-heat-inactivated sample was centrifuged, and the supernatant put in triplicates in a 96-well plate. Bradford reagent was then added, and absorbance measurements taken at 600 nm.
3
Plasma Lipid and Leptin Analysis
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Blood was collected by retro-orbital puncture of anesthetized mice. Plasma levels of nonesterified FA, glycerol, and TAG were measured using the commercially available kits NEFAC (WAKO Chemicals, Germany), TG Infinity Reagent (Thermo Fisher Scientific, MA), and Free Glycerol Reagent (Merck, Darmstadt, Germany). Plasma leptin was determined using a mouse leptin ELISA (catalog no.: 90030; Crystal Chem, IL).
4
Glycerol Quantification Assay in Mouse VAT
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VAT obtained from mice was finely diced and transferred to wells of 24-well plates containing 1 ml of DMEM with 4.5 g/L d -glucose (Gibco, Life technology, Gaithersburg, MD), and cultured in a humidified atmosphere of 21% O2 and 5% CO2 at 37 C for 1 h as previously described17 (link),45 (link). After refreshing the medium, the tissues were incubated with or without increasing doses of Adm for 24 h and culture medium was collected for glycerol analysis. The glycerol level in culture medium was assessed using Free Glycerol Reagent (Sigma Aldrich, St. Louis, MO, USA) according to manufacture instructions. The absorbance at A540 were read and recorded by a Spectrophotometer CLARIO STAR (BMG Labtech, Inc., Cary, NC, USA) as previously described17 (link).
5
Lipofectamine-mediated Transfection and Silencing in Adipocytes
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For expression assays, 3T3‐L1 cells, human adipocytes, and HEK-293 AD cells, were transfected with the corresponding plasmid vectors at 2.5 µg/mL using a 7.5:1000 dilution of Lipofectamine 2000 (Invitrogen), and cultured for 48 h prior to the experiments. For silencing studies, cells were transfected with a 7.5:1000 dilution of Lipofetamine RNAiMAX (Invitrogen) and mouse Rab34 siRNA (Dharmacon), mouse UBA1 siRNA (Dharmacon), or control siRNA (Sigma-Aldrich) (scrambled-transfected cells) at 25 nmol/L. Then, cells were kept in culture for 72 h. At the end of the experiments, cells were processed for confocal microscopy and/or immunoblotting as indicated in the corresponding sections. In another set of experiments, cells were collected in radioimmunoprecipitation assay (RIPA) buffer and intracellular concentration of TGs was determined using Triglyceride Reagent (Sigma-Aldrich) and Amplex UltraRed Reagent (Invitrogen), while culture media were analyzed for free glycerol content using Amplex UltraRed Reagent (Invitrogen) and Free Glycerol Reagent (Sigma-Aldrich) as previously described [24 (link)].
6
Quantifying Cellular Triglyceride Content
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Triglycerides were isolated with Hexan/Isopropanol (3:2). Triglyceride Reagent, Free Glycerol Reagent, and Glyerol Standard (all from Sigma-Aldrich, Munich, Germany) was used to measure cellular triglyceride content of adipocytes according to the manufacturer’s instructions.
7
Adipose Tissue Lipolysis Assay
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Subcutaneous adipose tissue (SAT), epididymal adipose tissue (EAT), and BAT were excised from 8-week-old male cd47-deficient and WT littermate control mice after a 6-h fast. Tissue was weighed and kept in ice cold PBS until all tissue was collected. Adipose tissue (80–180 mg) was placed in one well of a 24-well plate (n=3 mice/group; all tissue samples from each mouse were triplicated). Tissues were serum starved in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 1% fatty acid-free bovine serum albumin (BSA) for 1 h at room temperature (RT). Tissue was then treated with Krebs Ringer Buffer (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 2.6 mM MgSO4, 5 mM HEPES, pH adjusted to 7.2) in the presence of vehicle or isoproterenol (10 µM, Sigma-Aldrich, St. Louis, MO, USA) as previously described (Guo et al., 2013 (link)). After 2 h, media was collected to measure glycerol release with the Free Glycerol Reagent and appropriate standards (Sigma-Aldrich, St. Louis, MO, USA). Values were normalized to gram tissue weight (n=3 animals/group and n=3 experimental replicates/animal).
8
Adipose Tissue Glycerol Release Assay
9
Analysis of Lipolysis Signaling Pathway
10
Triglyceride Quantification in Milk Lipids
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