The cultured medium was collected to determine the extracellular glycerol content using a Free Glycerol Assay kit (ab65337, Abcam, Cambridge, United Kingdom) according to the protocol for the colorimetric assay provided by the manufacturer. Briefly, 2 × 106 snap frozen cells (−80 °C) were resuspended and homogenised in 500 μL of ice-cold glycerol buffer. After centrifugation (15 min at 4 °C at 10,000× g), the supernatant was collected. Several dilutions of the sample were performed using the Glycerol Assay Buffer. A standard curve was prepared from 1 mM Glycerol Standard provided in the kit. Samples and the standard curve were mixed with Reaction Mix (containing a glycerol probe, glycerol enzyme mix, and Glycerol Assay Buffer) in a clean 96-well plate. Plates were incubated at 37 °C for 30 min and protected from light. In the assay, glycerol is enzymatically oxidised to generate a product that reacts with the probe to generate colour. Finally, absorbance was determined spectrophotometrically at 570 nm (ASYS UVM 340, Cambridge, United Kingdom, Microplate Readers).
Free glycerol assay kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Free Glycerol Assay Kit is a colorimetric assay that quantifies free glycerol in biological samples. The kit provides the necessary reagents and protocols to perform the assay.
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Lab products found in correlation
Free fatty acid quantification kit, by Abcam (2 mentions)
Sunrise microplate reader, by Tecan (1 mentions)
Ab65336, by Abcam (1 mentions)
Mouse leptin elisa kit, by Crystal Chem (1 mentions)
Labassay nefa kit, by Fujifilm (1 mentions)
Cell proliferation kit 1, by Merck Group (1 mentions)
Camp enzyme immunoassay kit, by Merck Group (1 mentions)
Pka kinase activity assay kit, by Abcam (1 mentions)
Camp assay kit, by Abcam (1 mentions)
Cytotoxicity ldh assay kit wst, by Dojindo Laboratories (1 mentions)
Triglyceride assay kit, by Abcam (1 mentions)
Human total angiotensinogen assay kit, by Immuno-Biological Laboratories (1 mentions)
Crystal violet staining solution, by Merck Group (1 mentions)
Labassay triglyceride kit, by Fujifilm (1 mentions)
Gpdh assay kit, by Cosmo Bio (1 mentions)
Free fatty acid assay kit, by Abcam (1 mentions)
Forskolin, by Merck Group (1 mentions)
Triglyceride quantification kit, by Abcam (1 mentions)
Celltiter blue cell viability assay, by Promega (1 mentions)
Epichlorohydrin, by Merck Group (1 mentions)
17 protocols using free glycerol assay kit
1
Extracellular Glycerol Quantification Protocol
2
Lipolysis and cAMP Measurement in Adipose Tissue
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Fat pads were harvested and cut into small pieces, which were weighed and incubated in 600 μl of lipolysis medium (Gibco® DMEM 21063, Thermo Fisher Scientific, Waltham, MA, USA; supplemented with 2% BSA) either without additive or 10 μM isoproterenol. After 1 h of incubation at 37 °C, medium was collected, followed by determination of the glycerol concentration using a free glycerol assay kit (Abcam, Cambridge, UK), according to the manufacturer's instructions. Sample absorbance was measured at 570 nm using the Tecan Sunrise microplate reader (Tecan, Männedorf, Switzerland). Glycerol content was normalized to the initial tissue weight.
For cAMP measurement, fat pads were treated in an analogous manner and incubated in medium additionally supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX) and containing the respective additives for 15 min. Adipose tissue pieces were collected after incubation and frozen in liquid nitrogen. Subsequently, the intracellular content of cAMP was detected with a cAMP competitive enzyme immunoassay (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.
For cAMP measurement, fat pads were treated in an analogous manner and incubated in medium additionally supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX) and containing the respective additives for 15 min. Adipose tissue pieces were collected after incubation and frozen in liquid nitrogen. Subsequently, the intracellular content of cAMP was detected with a cAMP competitive enzyme immunoassay (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.
3
Quantifying Cellular Lipolysis
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The level of steatolysis was determined by measuring the release of free glycerol from cells or mouse serum. Glycerol content in cell culture medium and serum was determined using the Free Glycerol Assay Kit (ab65337; Abcam) according to the manufacturer’s instructions.
4
Quantification of Intracellular Triglycerides
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Intracellular TG content was determined by a quantification assay kit (ab65336, abcam®, UK). Briefly, cells were resuspended in 5% NP-40/ddH2O solution and heated at 90-100°C to solubilize TG. The cultured medium was collected to analyze the extracellular glycerol content using Free Glycerol Assay kit (ab65337, abcam®, UK). Determinations of intracellular TG and extracellular glycerol contents were carried out according to the protocols provided by the manufacturer.
5
Metabolic Response to K1 Toxin
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BY4742 cells were cultivated in JG medium until OD600 0.5 was reached and subsequently incubated with 1,000 AU K1 toxin concentrate. Samples were taken over 5 h, and directly quenched in 60% methanol (-40°C); extraction was performed as described by Sasidharan et al. (2012) (link). Briefly, cell-free supernatant was collected after short centrifugation and lyophilized (extracellular). Intracellular metabolite extraction was performed via cell lysis using methanol-chloroform and glass beads. Samples were centrifuged, the aqueous layer was recovered (intracellular), and also lyophilized. ATP measurement was conducted using the luminescence-based “ATP Determination Kit” (Thermo Fisher), Glycerol levels were determined using the “Free Glycerol Assay Kit” (Abcam). All samples were prepared in triplicates and kits were used according to the instructions of the manufacturer.
6
Mouse Plasma Metabolite Quantification
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Twenty mice were divided into five groups: 1 (2 h, cold), 2 (4 h, cold), 3 (2 h, thermoneutral), 4 (4 h, thermoneutral) and 5 (home cage, 0 h). Quantification of blood plasma measurement was performed according to the manufacturers’ recommendations: Mouse Leptin ELISA kit (Crystal Chem, no. 90030), Free fatty acid quantification kit (abcam, no. ab65341) and Free glycerol assay kit (abcam, no. ab65337).
7
Serum Free Fatty Acid and Glycerol Assay
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Blood samples were collected from the heart. Serum levels of free fatty acid and glycerol were measured with LabAssay NEFA Kit (294‐63601, Wako) and Free Glycerol Assay Kit (ab65337, Abcam), respectively, according to the manufacture's protocols.
8
Comprehensive Cell Signaling Analysis
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The Cell Proliferation Kit I (#11465007001, Sigma-Aldrich, St. Louis, MO, USA), Free Glycerol Assay Kit (ab65337, Abcam, Cambridge, MA, USA), cAMP Enzyme Immunoassay Kit (CA200, Sigma-Aldrich), and PKA Kinase Activity Assay Kit (ab139435, Abcam) were used to measure cell proliferation, glycerol content in culture medium, intracellular cAMP concentration, and intracellular PKA activity, respectively, according to the instructional guides provided by the manufacturers.
9
Assessing Metabolic Changes in ciBA Cells
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Cell culture supernatants and extracted cellular lysates were collected from the control fibroblasts, ciBAs, and ciBAs treated with Ang II, telmisartan, or Iso. The amounts of free glycerol and triglycerides were measured using Free Glycerol Assay Kit (ab65337, Abcam) and Triglyceride Assay kit (Ab65336, Abcam), respectively, according to the manufacturer’s instructions. cAMP levels in each ciBA sample were measured using a competitive ELISA method (cAMP Assay Kit, ab65355, Abcam). The secretion of AGT from ciBAs into the supernatant was quantified using a sandwich ELISA (Human Total Angiotensinogen Assay Kit, Immuno-Biological Laboratories, Gunma, Japan). To evaluate the cytotoxicity of Ang II treatment, the activity of lactate dehydrogenase (LDH) released into the culture supernatants was measured using Cytotoxicity LDH Assay Kit-WST (Dojindo) according to the manufacturer’s instructions. Glycerol, triglycerides, cAMP, and AGT levels were normalised to the total amounts of protein in each cell culture. All the experiments were performed in triplicates. Experiments were independently performed at least twice.
10
Glycerol Release Assay for Adipogenesis
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To perform the glycerol release assay, at the terminal differentiation time (PID 9), cells were incubated in culture medium without FBS for 24 h. After that, the supernatant was collected, and glycerol content measured according to manufacturer’s instructions (Free Glycerol Assay Kit; ab65337, Abcam, Cambridge, UK). Glycerol content was normalized to cell viability measure using crystal violet staining solution (Merck, Darmstadt, Germany) as previously described [58 (link)].
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