Apolipoprotein B Secretion Assay from Primary Hepatocytes
初级肝细胞中载脂蛋白B分泌测定
发布: 2024年05月05日第14卷第9期 DOI: 10.21769/BioProtoc.4982 浏览次数: 988
评审: Ralph Thomas BoettcherKrishna NakuluriAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2023
Abstract
Apolipoprotein B (APOB) is the primary structural protein of atherogenic lipoproteins, which drive atherogenesis and thereby lead to deadly cardiovascular diseases (CVDs). Plasma levels of APOB-containing lipoproteins are tightly modulated by LDL receptor–mediated endocytic trafficking and cargo receptor–initiated exocytic route; the latter is much less well understood. This protocol aims to present an uncomplicated yet effective method for detecting APOB/lipoprotein secretion. We perform primary mouse hepatocyte isolation and culture coupled with well-established techniques such as immunoblotting for highly sensitive, specific, and semi-quantitative analysis of the lipoprotein secretion process. Its inherent simplicity facilitates ease of operation, rendering it a valuable tool widely utilized to explore the intricate landscape of cellular lipid metabolism and unravel the mechanistic complexities underlying lipoprotein-related diseases.
Key features
• A pipeline for the isolation and subsequent culture of mouse primary hepatocytes.
• A procedure for tracking the secretion of APOB-containing lipoproteins.
• A rapid and sensitive assay for detecting the APOB level based on immunoblotting.
Keywords: Cardiovascular diseases (心血管疾病)
Hyperlipidemia (高脂血症)
Lipoprotein secretion (脂蛋白分泌)
Primary hepatocytes (初级肝细胞)
Western blot (蛋白质印记法)
Background
Cardiovascular diseases (CVDs) continue to be the leading cause of death worldwide, accounting for over 20 million deaths each year. Hyperlipidemia, characterized by elevated plasma levels of cholesterol and triglycerides, is the most common risk factor for CVDs [1–3]. Due to their hydrophobic nature, lipids are loaded onto specialized carriers named lipoproteins before entering the circulation to meet the lipid demands of various body parts. Excess plasma lipids, especially those carried by apolipoprotein B (APOB)-containing lipoproteins, are the major cause of plaque buildup on the arteries, which leads to narrowed blood vessels, obstruction of blood flow, and ultimately CVDs. The APOB is the primary and unexchangeable structural protein of atherogenic lipoproteins, including low-density lipoproteins (LDL) and chylomicrons (CM), originating from hepatocytes and enterocytes, respectively [4–6]. Stoichiometrically, each APOB-containing lipoprotein contains one APOB molecule, which renders the plasma APOB levels a more reliable risk indicator for CVDs than plasma lipid levels per se.
Understanding the secretion process of lipoproteins is crucial for unraveling their roles in hyperlipidemia and CVDs. Abnormalities in lipoprotein secretion may result in elevated blood lipoprotein levels, further contributing to the progression of atherosclerosis. Therefore, developing an efficient and accurate method for detecting lipoprotein secretion is essential for a profound understanding of their biological functions and roles in disease development [7–11]. Here, we report a straightforward approach including the isolation and culture of primary mouse hepatocytes, followed by detecting the secreted APOB in the medium using established techniques like immunoblotting. Enzyme-linked immunosorbent assay (ELISA) can also be used for APOB secretion measurement, and the above methods are compatible. Our method provides a reliable, sensitive, specific, and semi-quantitative strategy for lipoprotein secretion detection, which can be feasibly applied to explore cellular lipid metabolism. This method may contribute to advancing the understanding of the regulatory mechanisms of lipoprotein export itinerary and holds promise for providing new insights into the prevention and treatment of CVDs.
Materials and reagents
25 mg/mL IV collagenase (Sigma-Aldrich, catalog number: C5138)
DMEM culture medium (Gibco, catalog number: 8123306)
Penicillin-Streptomycin solution (P/S), 100× (Caisson, catalog number: PSL01)
Phosphate buffered saline (PBS) (Meilunbio, catalog number: MA0015)
Fetal bovine serum (FBS) (Vistech, catalog number: SE100-011)
Acetone (TGREAG, catalog number: 105003)
APOB polyclonal antibody (Proteintech, catalog number: 20578-1-AP)
ALBUMIN monoclonal antibody (Proteintech, catalog number: 66051-1-Ig)
Glucose (HARVEYBIO, catalog number: SR1742)
NaHCO3 (TGREAG, catalog number: 114005)
NaCl (TGREAG, catalog number: 112008)
HEPES (Sigma, catalog number: V900477)
KCl (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10016328)
KH2PO4 (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10017628)
MgSO4·7H2O (Sinopharm Chemical Reagent Co., Ltd, catalog number: 10013018)
Tris-HCl (Sigma, catalog number: V900483)
Nonidet P-40 (Sigma, catalog number: 56741)
Glycerol (Sigma, catalog number: G7893)
EGTA (Sigma, catalog number: 03777)
CaCl2 (TGREAG, catalog number: 104034)
Solutions
Krebs Ringer with glucose (KRG) buffer (see Recipes)
Solution C (see Recipes)
Buffer 1 (see Recipes)
Buffer 2 (see Recipes)
Lysis buffer (see Recipes)
Recipes
KRG buffer (1 L)
Note: KRG buffer (pH = 7.4) should be prepared immediately before use. Filter through a 0.22 μm membrane before use.
Reagent Final concentration Quantity or Volume Glucose 20 mM 3.6 g NaHCO3 25 mM 2.0 g NaCl 120 mM 7.0 g HEPES 1 M (pH 7.45) 5 mL Solution C (Recipe 2) N/A 10 mL Total N/A Set volume to 1 L with ddH2O Solution C (1 L)
Reagent Final concentration Quantity or Volume KCl 480 mM 35.79 g KH2PO4 120 mM 16.33 g MgSO4·7H2O 120 mM 29.58 g Total N/A Set volume to 1 L with ddH2O Buffer 1
Add 100 μL of 50 mM EGTA to 50 mL of KRG buffer.
Buffer 2
Add 100 μL of 1 M CaCl2 and 25 mg of IV collagenase to 50 mL of KRG buffer.
Lysis buffer
Note: Store at 4 °C and add protease inhibitor before use.
Reagent Final concentration Tris-HCl (pH 7.4) 50 mM Nonidet P-40 1% Glycerol 10% NaCl 150 mM
Laboratory supplies
Cell strainer (Falcon, catalog number: 352350)
Millex-GP Filter, 0.22 μm, PES 33 mm, non-sterile (Millex, catalog number: SLGP033NS)
Axygen MaxyClear Snaplock microtubes (Axygen, catalog number: MCT-150-C)
10 cm cell culture dish (JET, catalog number: TCD010100)
6-well cell culture dish (JET, catalog number: TCP010006)
50 mL centrifuge tube (JET, catalog number: CFT011500)
10 mL pipettes (JET, catalog number: GSP010010)
Perfusion set (BD Intima II, catalog number: 383405; TNTC-1066)
Eye scissors (Beijing Hongbai Technology Co., Ltd., catalog number: 2101157)
Forceps (Beijing Hongbai Technology Co., Ltd., catalog number: HC10704)
Nitrocellulose membrane (DiNing, catalog number: 66485)
Equipment
Centrifuge (Eppendorf, model: 5424R)
Centrifuge (Eppendorf, model: 5810)
GE Amersham Imager600 (GE Healthcare, catalog number: 29083463)
Western blot equipment (Bio-Rad, catalog number: 1658033)
Procedure
文章信息
版权信息
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Wang, Y., Li, X., Huang, R., Chen, X. W. and Wang, X. (2024). Apolipoprotein B Secretion Assay from Primary Hepatocytes. Bio-protocol 14(9): e4982. DOI: 10.21769/BioProtoc.4982.
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分类
细胞生物学 > 基于细胞的分析方法 > 蛋白质分泌
医学 > 心血管疾病
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