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.
E1005
Manufactured by Applygen
Sourced in China
The E1005 is a versatile laboratory equipment designed for general scientific use. It functions as a precision measurement device, capable of accurately recording and displaying various data points. The core purpose of the E1005 is to provide reliable and consistent data collection for research and analytical applications.
Automatically generated - may contain errors
9 protocols using e1005
1
Biochemical Analysis of Blood Plasma
2
Cholesterol Measurement in Plasma and Liver
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3
Quantification of Plasma Lipoproteins by FPLC
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Serum total cholesterol and LDL cholesterol levels were determined using commercial assay kits (#E1005 and #E1018, respectively, from Applygen, Beijing, China). Fast protein liquid chromatography (FPLC) analysis of plasma lipoproteins was performed as described (Guo et al, 2018 (link)). Briefly, pooled plasma aliquots (200 μl from each group) were applied to Tricorn high‐performance Superose S‐6 10/300GL columns and filtered using an Amersham FPLC system (GE Healthcare, Chicago, IL, USA), followed by elution with PBS at a constant flow rate of 0.25 ml/min. Eluted fractions (500 μl per fraction) were assessed for cholesterol concentrations using enzymatic assay (Guo et al, 2018 (link)). FPLC was carried out in an investigator‐blind manner.
4
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).
5
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.
6
Quantifying Hepatic Cholesterol Content
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Plasma and hepatic total cholesterol (Tch ) contents were measured using commercial assay kits (E1005 and E1015) from Applygen Technologies Inc. (Beijing, China), according to the kits instructions. Briefly, 50 mg ground liver samples were homogenized in 1 mL lysis buffer supplied in the kit by a homogenizer (Hoder, Nanjing, China). Then, the homogenates were stood for 10 min and heated in water bath for 10 min at 70°C. Centrifugation was performed at a speed of 2,000 rpm for a duration of 5 min. The liquid above the sediment was gathered and the Tch concentrations were determined by microplate reader (Tecan, Männedorf, Switzerland).
7
Metabolic Profiling of DLK1-Deficient Mice
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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).
8
Serum Lipid Profile Measurement
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Serum concentration of total cholesterol was measured using a commercial cholesterol assay kit (E1005, Applygen Technologies, Inc. Beijing, China). Serum concentrations of high-density lipoprotein-cholesterol (HDL-C , 0063280, Beijing BHKT Clinical Reagent Co., China) and low-density lipoprotein-cholesterol (LDL-C , 006340, Beijing BHKT Clinical Reagent Co., China) were measured with respective assay kits.
9
Cholesterol and Bile Acid Quantification
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Total hepatic cholesterol concentration was detected by a commercial total cholesterol assay kit (#E1005; Applygen-Technologies, Inc., Beijing, China). Total bile acid concentrations were measured using enzymatic colorimetric methods with a commercial kit (E003; Nanjing Jian-cheng Bio-engineering Institute, Nanjing, China).
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