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      Potential Applications of Fluorescence-Activated Cell Sorting (FACS) and Droplet-Based Microfluidics in Promoting the Discovery of Specific Antibodies for Characterizations of Fish Immune Cells

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          Abstract

          Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.

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          Microfluidics: Fluid physics at the nanoliter scale

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            Continuous cultures of fused cells secreting antibody of predefined specificity.

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              mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish.

              Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                16 November 2021
                2021
                : 12
                : 771231
                Affiliations
                [1] 1 State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University , Ningbo, China
                [2] 2 Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University , Ningbo, China
                [3] 3 Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University , Ningbo, China
                Author notes

                Edited by: Jia Cai, Guangdong Ocean University, China

                Reviewed by: Teruyuki Nakanishi, Goto Aquaculture Research Institute, Japan; Jianmin Ye, South China Normal University, China

                *Correspondence: Jiong Chen, jchen1975@ 123456163.com

                This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology

                Article
                10.3389/fimmu.2021.771231
                8635192
                34868030
                46562088-76e7-41f6-bfc7-82021156aecc
                Copyright © 2021 Fei, Nie, Zhang and Chen

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 06 September 2021
                : 21 October 2021
                Page count
                Figures: 3, Tables: 1, Equations: 0, References: 151, Pages: 15, Words: 5512
                Funding
                Funded by: Natural Science Foundation of Zhejiang Province , doi 10.13039/501100004731;
                Funded by: National Natural Science Foundation of China , doi 10.13039/501100001809;
                Funded by: Natural Science Foundation of Ningbo , doi 10.13039/100007834;
                Categories
                Immunology
                Review

                Immunology
                teleost fish,immune cells,monoclonal antibodies,fluorescence-activated cell sorting,droplet-based microfluidics

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