The detection of lipolysis rate was performed as previously described by Berger and Barnard [19 (link)]. Briefly, the samples of adipose tissues (0.2 g) were minced, and then incubated in 2 mL of 25 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (pH 7.4) containing 1 μMisoproterenol at 37 °C. The glycerol levels were determined by a glycerol assay kit (Randox Laboratories) after 1, 2, and 3 h of incubation. The absorbance at 520 nm was measured by a Hitachi U2800A spectrophotometer. The equation of micromoles of glycerol released per gram of adipose tissue per hour was used to indicate the lipolysis rate.
Glycerol assay kit
Manufactured by Randox
Sourced in United Kingdom
The Glycerol assay kit is a laboratory product manufactured by Randox. It is designed to quantitatively determine the concentration of glycerol in a sample.
Automatically generated - may contain errors
Lab products found in correlation
U2800a spectrophotometer, by Hitachi (2 mentions)
Cholestyramine, by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by PerkinElmer (1 mentions)
Cholic acid, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Ampk and phospho ampk thr 172, by Cell Signaling Technology (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
4 protocols using glycerol assay kit
1
Adipose Tissue Lipolysis Quantification
2
Biochemical Analysis of Metabolic Pathways
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Cholesterol, cholic acid, heparin, and cholestyramine were obtained from Sigma–Aldrich (St. Louis, MO, USA). The enzymatic assay kits for detection of TC and TG were provided by Audit Diagnostics (Cork, Ireland). The enzymatic assay kits for detection of AST and ALT were obtained from Randox Laboratories (Antrim, UK). A bile acid assay kit was purchased from Randox Laboratories. A glycerol assay kit was purchased from Randox Laboratories. Hematoxylin and eosin staining solution were obtained from Leica Biosystems (Richmond, IL, USA). RIPA lysis and extraction buffer was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene difluoride membranes were obtained from Bio-Rad Laboratories (Hercules, CA, USA). An enhanced chemiluminescence detection kit was obtained from PerkinElmer (Waltham, MA, USA). The antibodies for AMPK and phospho-AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies for farnesoid X receptor (FXR), peroxisome proliferator activated receptor (PPAR)-α, PPAR-γ, low density lipoprotein receptor (LDLR), cytochrome P450-7A1 (CYP7A1), and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
3
Lipolysis and Lipase Activity Assays
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The detection of lipolysis rate was performed as previously described by Berger and Barnard [41 (link)]. Briefly, samples (0.2 g perirenal adipose tissues) were minced, and then mixed in 2 mL of 25 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (pH 7.4) containing 1 μM isoproterenol at 37°C. A glycerol assay kit (Randox Laboratories, Antrim, UK) was used to measure the glycerol levels after 1, 2, and 3 h of incubation. A Hitachi U2800A spectrophotometer was used to detect the absorbance at 520 nm. An equation of micromoles of glycerol released per gram of adipose tissue per hour indicated the lipolysis rate.
The activity of LPL in the adipose tissues was analyzed as previously described by Kusunoki et al. [42 (link)]. Briefly, samples (0.1 g adipose tissues) were minced, and then mixed in Krebs–Ringer bicarbonate buffer (pH 7.4) containing 10 units/mL heparin for 60 min at 37 °C. The sample solution was then reacted with an equal volume of p-nitrophenyl butyrate (2 mM). A Hitachi U2800A spectrophotometer was used to measure the absorbance at 400 nm. The amount of p-nitrophenol formation over the 10 min incubation indicated the LPL activity.
The activity of LPL in the adipose tissues was analyzed as previously described by Kusunoki et al. [42 (link)]. Briefly, samples (0.1 g adipose tissues) were minced, and then mixed in Krebs–Ringer bicarbonate buffer (pH 7.4) containing 10 units/mL heparin for 60 min at 37 °C. The sample solution was then reacted with an equal volume of p-nitrophenyl butyrate (2 mM). A Hitachi U2800A spectrophotometer was used to measure the absorbance at 400 nm. The amount of p-nitrophenol formation over the 10 min incubation indicated the LPL activity.
4
Quantifying Adipose Tissue Lipolysis
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The lipolysis rate was detected as previously described [32 (link)]. Briefly, the minced adipose tissues were reacted in a working buffer containing N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (pH 7.4) and isoproterenol at 37 °C. After reaction, the product of glycerol was analyzed with a glycerol assay kit (Randox Laboratories, London, UK), and the absorbance at 520 nm was detected. The lipolysis rate is presented as μmol glycerol release per gram tissue per hour.
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