The same batch of DLK1−/− and wild-type mice were fasted for 12 h under free-drinking conditions. The next day, blood samples were collected from the posterior orbital venous plexus in 1.5 mL anticoagulant tubes containing potassium citrate and then centrifuged for 20 min at 3000 rpm at 4 °C. The supernatant was collected in a new 1.5 mL sterilized centrifuge tube for subsequent determination of plasma biochemical parameters. Plasma TG, CHOL, Glu, HDL and LDL levels were detected by Enzymic assay kit for total triglyceride, cholesterol, glucose, HDL, LDL in liquid samples (E1003, E1005, E1010, E1018 and E1017, Applygen Technologies, Beijing, China), which were used as described in the instructions. Then, the absorbance of the samples was detected using a multi-function microplate reader (Biotech, San Francisco, CA, USA).
E1003
Manufactured by Applygen
Sourced in China
The E1003 is a laboratory centrifuge designed for general-purpose applications. It is capable of separating and concentrating samples at high speeds. The device features a compact and durable construction, with a rotational speed range of up to 6,000 rpm. Detailed specifications and intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
E1013, by Applygen (5 mentions)
E1005, by Applygen (4 mentions)
E1010, by Applygen (1 mentions)
E1015, by Applygen (1 mentions)
Lysis buffer, by Sangon (1 mentions)
P1511, by Applygen (1 mentions)
Palmitate, by Sangon (1 mentions)
Insulin elisa kit, by Mercodia (1 mentions)
Adi 900 097, by Enzo Life Sciences (1 mentions)
10 protocols using e1003
1
Metabolic Profiling of DLK1-Deficient Mice
2
Comprehensive Lipid Profiling Protocol
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The content of total cholesterol and triglycerides (TG) in serum was measured using commercial assay kit (E1005 and E1003 respectively; Applygen Technologies, Inc. Beijing, China). Concentrations of LDL cholesterol (LDLC) and high-density lipoprotein cholesterol (HDLC) in serum were measured with respective assay kits (006340 and 006328, Beijing BHKT Clinical Reagent Co., Ltd., Beijing, China). The cholesterol and TG in liver was determined according to the instruction of a tissue commercial assay kit (E1015 and E1013 respectively; Applygen Technologies, Inc. Beijing, China). Hepatic and serum bile acid content was detected by enzymatic colorimetric methods according to a commercial bile acid assay kit (E003; Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).
3
Quantification of Triglyceride Levels
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The TAG content was detected as described by Ding et al. (2012) and Du et al. (2018b) . Briefly, TAG content in cells and supernatant was determined using commercial kits (E1003, E1013, Applygen Technologies Inc.). The cell-free supernatant (1 mL) in each group was collected following treatments. Hepatocytes were harvested and washed twice with ice-cold PBS. Subsequently, cells were lysed with lysis buffer (Sangon Biotech Co. Ltd.) in an ice bath. The lysate was centrifuged at 12,000 × g at 4°C for 5 min and the supernatant collected. A portion of the supernatant was used to determine total protein concentrations using the bicinchoninic acid assay (P1511, Applygen Technologies Inc.). The remaining supernatant was heated in a water bath (70°C) for 10 min, and centrifuged at 800 × g for 5 min at room temperature. A 10-μL supernatant was then mixed with 190 μL of chromogenic liquid before measuring TAG. A 30-μL sample from the cell-free supernatant was mixed with 170 μL of chromogenic liquid before measuring TAG assay. The absorbance at 550 nm is proportional to the concentration of TAG.
4
Modeling Hepatic Lipid Accumulation
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To establish cellular models of lipid accumulation, primary hepatocytes were seeded in six-well plates. After cell adhesion, 0.6 mM a mixture of free fatty acids (FFAs) was added to the medium for 24 hours (at a final ratio of 2:1 with oleate and palmitate from Sangon Biotech, China). The cells were then fixed with 4% paraformaldehyde for 10 minutes and stained with nile red and Hoechst staining for 10 minute to visualize intracellular lipid accumulation. The triglyceride (TG), total cholesterol (TC) contents in isolated primary hepatocytes or liver tissue were measured using the kits from Applygen, China (#E1003 and E1005, respectively), according to the manufacturers’ instructions.
5
Plasma and Hepatic Triglyceride Measurement
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The concentration of triglyceride (TG) in plasma were measured by an automatic biochemical analyzer (7020, HITACHI, Tokyo, Japan) using commercial kits (E1003; Applygen Technologies Inc., Beijing, China).Hepatic TG were measured using a triglyceride assay kit(E1013; Applygen Technologies Inc., Beijing, China) following the manufacturer's instructions.
6
Biochemical Analysis of Blood Plasma
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For biochemical analysis of the blood, samples were collected from tail vein blood and were transferred into EDTA-containing tubes. After centrifugation at 1500×g for 15 min at room temperature, the plasma was transferred into a new tube and stored at -80°C until use. The plasma levels of total cholesterol and triglyceride were measured using an ELISA kit (E1005 and E1003) from Applygen Technologies Inc. (Beijing, China). The plasma levels of BHB and acetoacetate (AcAc) were measured using an ELISA Kit (ab83390 and ab180875) purchased from Abcam (Cambridge, MA). Assays were performed according to the protocols provided by the manufacturer. After behavioral tests, the rats were anaesthetized and sacrificed by decapitation. The brains were rapidly removed, and the hippocampus was dissected and frozen at -80°C for future western blot analysis. The brains used for immunohistochemistry analysis were fixed in 4% formaldehyde immediately after isolation.
7
Biochemical Analysis of Mouse Plasma
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Mouse blood samples were collected via right ventricle with EDTA (1.8 mg/ml) after 5 h fasting and then centrifuged at 400 g at 4°C for 20 min to separate plasma. Plasma insulin concentration was measured by insulin ELISA kit (10-1247-01; Mercodia, Uppsala, Sweden) following the manufacturer’s instructions. Plasma triglyceride concentration was measured using a triglyceride measurement kit (E1003; Applygen Technologies Inc., Beijing, China). Plasma alanine transaminase (ALT) and aspartate transaminase (AST) activity was detected by ALT assay kit (C009-2; Njjcbio, Jiangsu, China) and AST assay kit (C010-2; Njjcbio), respectively.
8
Triglyceride Assay and Muscle Fiber Typing
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The triglyceride assay kit (Applygen Technologies Inc, E1003) was used to determine serum triglyceride levels. In the triglyceride assay protocol, triglycerides are converted to free fatty acids and glycerol. Glycerol is then oxidized to generate a product which reacts with a probe to generate color and fluorescence. The muscle fibers were stained with ATPase staining method (Solarbio, G2380). In brief, gastrocnemius muscle from mice was frozen in isopentane near its freezing point and made into 10-μm-thick frozen sections. Frozen sections were then incubated in acid preincubation solution (pH 4.2, 5 min), ATPase incubation solution (30 min), 1% CaCl2 solution (3 min), cobalt solution (3 min), and sulfide working solution (1 min). The frozen sections were then washed with water and decolorized with anhydrous ethanol. Slow muscle fiber type was determined by the ATPase-positive part (black precipitation).
9
Triglyceride Liver and Plasma Analysis
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10
Triglyceride and Corticosterone Quantification
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Triglyceride in plasma and liver was measured by using respectively commercial triglyceride assay kit (E1003 and E1013) purchased from Applygen Technologies Inc., China, following the manufacturer’s instructions. Plasma CORT concentration was measured using a commercial EIA kit (No. ADI-900–097, Enzo, USA) according to the instructions of the manufacturer.
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