The triglyceride, glycogen, lactic acid levels in the livers of 100-day-old male zebrafish were measured using the Triglyceride Assay Kit (Item No. A110-1-1), Liver/Muscle Glycogen Assay Kit (Item No. A043-1-1), Lactic Acid Assay Kit (Item No. A019-2-1) purchased from Nanjing Jiancheng Bioengineering Institute. The tail of the zebrafish was cut off after anesthesia, and blood was collected from the severed tail with a micropipette gun rinsed with heparin sodium. Then the zebrafish was dissected to collect liver tissue. Each parallel sample required about 15mg of liver tissue or 10ul of plasma for the corresponding indicator detection. At least 3 parallel samples are required for each indicator. The detection kit for lactic acid in plasma is consistent with the detection kit for lactic acid in liver. The plasma FFA levels were measured using the Nonesterified Free fatty acids assay kit (Item No. A042-2-1) purchased from Nanjing Jiancheng Bioengineering Institute. The samples were processed following the procedure provided by the manufacturer. Each parallel sample required 4 ul of plasma, and the experiment was repeated eight times.
Nonesterified free fatty acids assay kit
Manufactured by Nanjing Jiancheng
Sourced in China
The Nonesterified Free Fatty Acids Assay Kit is a laboratory product designed to quantify the concentration of nonesterified free fatty acids in various samples. The kit utilizes an enzymatic colorimetric method to determine the levels of these fatty acids.
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Lab products found in correlation
Triglyceride assay kit, by Nanjing Jiancheng (6 mentions)
Lactic acid assay kit, by Nanjing Jiancheng (3 mentions)
Total cholesterol assay kit, by Nanjing Jiancheng (2 mentions)
Liver muscle glycogen assay kit, by Nanjing Jiancheng (1 mentions)
Ins elisa kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Cytiva (1 mentions)
Total triglyceride assay kit, by Nanjing Jiancheng (1 mentions)
Triglyceride quantification kit, by Abcam (1 mentions)
Atp assay kit, by Nanjing Jiancheng (1 mentions)
Ca2 mg2 atpase assay kit, by Nanjing Jiancheng (1 mentions)
Na k atpase assay kit, by Nanjing Jiancheng (1 mentions)
Au640, by Olympus (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Malondialdehyde assay kit, by Nanjing Jiancheng (1 mentions)
Total protein quantitative assay kit, by Nanjing Jiancheng (1 mentions)
Albumin assay kit, by Nanjing Jiancheng (1 mentions)
Total antioxidant capacity assay kit, by Nanjing Jiancheng (1 mentions)
Triglyceride reagent kit, by Nanjing Jiancheng (1 mentions)
15 protocols using nonesterified free fatty acids assay kit
1
Metabolic Profiling of Aged Zebrafish Liver
2
Measuring Metabolic Markers in Plasma
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Blood was collected from the left cardiac cavity and centrifuged at 3,000 × g for 10 min at 4°C to isolate the supernatant/plasma. FPG levels were determined using a glucose oxidase kit according to the manufacturer's protocol. The plasma levels of the fasting insulin were measured by INS ELISA kit (Thermo Fisher Scientific, Inc., cat. no. ERINS). Non-esterified free fatty acids assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. A042-2-1), total cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. F002-1-1) and triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, cat. no. A110-1-1) were used to measure FFA, TC and TG, respectively. The ISI was defined as follows: ISI=l g (1/FPG × serum insulin). The IR index was defined as follows: IR index=FPG level (mg/dl) × fasting serum insulin level (ng/ml)/22.5 (16 (link)).
3
Lipid Droplet Formation in CBRH-7919 and BRL3A Cells
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CBRH-7919 cells were maintained in RPMI-1640 medium (Hyclone Laboratories, South Logan, UT, USA) containing 10% FBS (GIBCO, Gaithersburg, MD, USA). BRL3A cells were maintained in DMEM (GIBCO, Gaithersburg, MD, USA) containing 10% FBS (GIBCO, Gaithersburg, MD, USA). For lipid droplet formation and oil red O staining, CBRH-7919 cells and BRL3A cells were plated in a 12-well plate. After 24 h, cells were treated with mixed FFAs (1 mM; PA:OA, 1:2 in RPMI-1640 medium containing 2% FBS) for the indicated time. Then, oil red O staining was conducted to determine the cellular lipid droplet formation. Treated cells were visualized using microscopy (Olympus, Tokyo, Japan). TG and FFA levels of cells and rat livers were measured, respectively, by using the Total Triglyceride Assay Kit (Nanjing Jiancheng, Nanjing, China) or the Non-esterified Free Fatty Acids Assay Kit (Nanjing Jiancheng, Nanjing, China), following the manufacturer’s instructions.
4
Measuring Lung Cytokines and Fatty Acids
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Lungs were harvested and homogenized using tissue homogenizer. Bronchoalveolar lavage fluid (BALF) was performed by cannulating the trachea with a blunt 22-gauge needle and then lavaging the lungs 3 times with 1 ml of ice-cold PBS. The levels of the TNF-α, IL-6 and interferon β (IFNβ) cytokines in the lung homogenates, BALF supernatant and cell culture supernatant were assayed using commercial ELISA (enzyme linked immunosorbent assay) kits according to the manufacturer’s instructions (Cloud-Clone Corp, China). Lung homogenates were also assayed for free fatty acids using Nonesterified Free Fatty Acids assay kit (Nanjing Jiancheng Bioengineering, China) according to the manufacturer’s protocol.
5
Liver Biochemical Marker Quantification
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The levels of serum alanine transaminase (ALT) and aspartate transaminase (AST) were measured using an automatic biochemistry analyzer (ARCHITECT c8000) following the manufacturer’s instructions. The levels of serum NEFA were measured by the Non-Esterified Free Fatty Acids Assay Kit (Jiancheng, People’s Republic of China). For hepatic triglyceride (TG) content measurement, 100 mg of liver was homogenized to release the lipid, which was further extracted by the Triglyceride Quantification Kit (BioVision, Inc., Milpitas, CA, USA). Total lipid was suspended in 5% NP-40 in water, and TG concentrations were then measured according to the manufacturer’s procedures.9 (link)
6
Metabolic Biomarker Quantification Protocol
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Nanjing Jiancheng Bioengineering Institute produced the ATP Assay Kit (#A095-1-1), Nonesterified Free Fatty Acids Assay Kit (#A042-2-1), and Triglyceride (TG) Assay Kit (#A110-1-1). Human Fatty Acid Oxidase (FAO) ELISA Kit (#JL48747) and Human Acetyl-Coenzyme A (Acetyl-CoA) ELISA Kit (#JL32777) were purchased from Jianglaibio (Shanghai, China). These kits were used to test adenosine triphosphate (ATP), FAO, TG, nonesterified fatty acid (NEFA), and Acetyl-CoA in cells. Each step strictly follows the instructions of the manual. Finally, the light signal at the specified wavelength was detected with a microplate analyzer [21 (link)].
7
Membrane ATPase Activity Assays
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The serum lactate levels, FFA concentrations, sodium-potassium-ATPase (Na+-K+-ATPase) activities, calcium-magnesium-ATPase (Ca2+-Mg2+-ATPase) activities, and the erythrocyte membrane ATPase activities were detected by colorimetry. Lactic acid assay kit, Non-esterified free fatty acids assay kit, Na+-K+-ATPase assay kit, and Ca2+-Mg2+-ATPase assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The experimental steps were in strict accordance with the manufacturer's instructions.
8
Metabolic Function Evaluation Protocol
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The creatinine was determined by the Creatinine (Cr) Assay Kit. Urine microalbumin was quantified by the Mouse Microalbumin ELISA Kit. The microalbumin to creatinine ratio (ACR) was determined by the ratio of the urinary microalbumin to the urinary creatinine. Creatinine clearance rate (Ccr) was calculated by the equation: Ccr = (urine creatinine × urine volume/24 h)/serum creatinine. The renal index (RI) was determined by the ratio of kidney weight to body weight, it is an objective, reproducible tool to predict renal function [46 (link)]. Postprandial blood glucose levels were measured once every 2 weeks employing the Glucose Assay Kit. Intraperitoneal glucose tolerance test (IPGTT) was performed after the mice fasted for 12 h. The serum triglyceride and free fatty acids (FFAs) were also measured by the Triglyceride Assay Kit and Nonesterified Free Fatty Acids Assay Kit (Nanjing Jiancheng Bioengineering, Nanjing, China).
9
Serum Lipid and Hormone Analysis in Rats
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After 6 months, rats were fasted for 12 h and anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). Blood (~10 ml) was collected in tubes and serum was separated by centrifuging the samples at 1,000 × g for 10 min at room temperature and stored at −80°C prior to biochemical analysis. Fasting TG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were measured using an automatic biochemical analyzer (AU640; Olympus Corporation, Tokyo, Japan). The serum levels of E2 were determined by radioimmunoassay (Beijing North Institute of Biological Technology, Beijing, China) and the serum levels of free FA (FFA) were measured using a Non-esterified Free Fatty Acids Assay kit (Nanjing Jiancheng Bio-Engineering Institute Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Serum PA levels were examined by gas chromatography-mass spectrometry according to the method of Han et al (22 (link)).
10
NRCM Protein Quantification and FFA Analysis
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After being harvested, NRCMs were disrupted using Manual cell disruptor, and then quantified according to Pierce® BCA protein assay kit (Thermo, Waltham, MA, USA). Free fatty acid content was detected using Nonesterified free fatty acids assay kit (Nanjing Jiancheng Bioengineering Institute, China).
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