Multiple approaches for the evaluation of connexin-43 expression and function in macrophages

IF 1.6 4区 医学 Q4 BIOCHEMICAL RESEARCH METHODS
Júlia Costa de Sousa , Stephanie Alexia Cristina Silva Santos , Eleonora Kurtenbach
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引用次数: 0

Abstract

Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions.

评估巨噬细胞中连接蛋白-43表达和功能的多种方法。
连接蛋白是重要的缝隙连接蛋白,在哺乳动物各种器官的细胞间通讯中发挥着关键作用。连接蛋白-43(Cx43)在免疫系统的各种成分中都有表达,有大量证据表明它参与了炎症反应。Cx43 参与巨噬细胞功能涉及嘌呤能信号通路。巨噬细胞有助于抵御细菌败血症和腹膜炎等炎症反应。有几种检测方法可以确定巨噬细胞中 Cx43 的存在和活性。实时聚合酶链反应(PCR)可测量 Cx43 的相对 mRNA 表达,而 Western 印迹法可检测蛋白质表达水平。利用免疫荧光测定法,可以分析 Cx43 在细胞或组织中的表达并观察其定位。此外,还可利用荧光染料显微注射或刮片装载-染料转移等功能测试来评估连接蛋白介导的细胞间隙连接通讯。选择性抑制剂的使用有助于加深对这一问题的理解,并强化了连接蛋白在各种过程中的作用。在此,我们将讨论这些评估 Cx43 和巨噬细胞间隙连接的方法。
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来源期刊
CiteScore
4.10
自引率
0.00%
发文量
120
审稿时长
3 months
期刊介绍: The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.
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