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

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.

A comprehensive overview of recent advances in generative models for antibodies

“…By adding substrates for colorimetric reactions and measuring enzyme activity, the concentration of antigens or antibodies in the sample can be quantified through standard curves [116] . FACS uses fluorescently labeled antibodies to bind to targets on the cell surface, and then sorts these cells using flow cytometry [117] . Detection of aggregates: In vivo, aggregates can be recognized by the immune system as foreign entities, leading to immunogenicity and adverse reactions.…”

Single cell genomics as a transformative approach for aquaculture research and innovation

“…A subset of genes will represent targets for which to develop monoclonal antibodies targeting candidate cell surface markers, which can subsequently be used to sort or quantify cells using fluorescence‐activated cell sorting (FACS); particularly useful in immunology (Section 3.2 ) (reviewed in Ref. 51 ). Finally, data generated by single cell transcriptomics can be used to deconvolute cell‐specific expression signals in bulk studies (e.g., Refs., 52 , 53 , 54 ).…”

Advances in application of single-cell RNA sequencing in cardiovascular research

“…The single-cell capture method varies with the cell volume. Mainstream single-cell capture methods include laser capture, fluorescence-activated cell sorting (FACS), microfluidic capture, and microdroplet-based capture (16)(17)(18). FACS uses the principle of light scattering and fluorescence signals to sort cells into 96-or 384-well plates.…”