In 1953, Crosland-Taylor of Middlesex Hospital published an article in Nature, introducing fluid mechanics focus to the field of cell detection (this principle, also known as sheath flow), which was the basis of flow cytometry. About 20 years later, the first commercial flow cytometer hit the market. Over the next 50 years, flow cytometry has been widely used in basic research and clinical diagnosis.
Applications of flow cytometry
One. Cell phenotypic assays
Immune cell phenotyping is the most prominent application of flow cytometry. Identify and characterize immune cell populations by detecting surface or intracellular markers. Flow cytometry enables precise identification and classification of immune cell populations such as T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, platelets, and granulocytes.
Immune cell-related phenotypic markers
T cell markers
Macrophage markers
Neutrophil markers
Fibroblast markers
DC cell markers
NK cell markers
Myeloid-derived suppressor cells (MDSCs) markers
B cell markers
Researchers can identify and quantify various immune cell subsets in heterogeneous populations. Clinicians can diagnose and monitor a variety of hematologic diseases, and perform immunoimmune assessments (8 categories of immune cell composition and tumor prognosis).
Two. Cell viability assays
Flow cytometry enables quantitative measurement of viable and non-viable cells cultured both within and in vitro. By using fluorescent dyes that selectively label live or dead cells, flow cytometry can provide accurate and reliable viability assays that help determine the percentage of cell viability.
1980年使用H-33342检测死细胞和活细胞（Cytometry 1980）
Flow cytometry can distinguish between live, apoptotic, and necrotic cells based on specific markers or dyes, providing a more detailed understanding of cell health and status. By combining viability dyes with markers for cell surface antigens, intracellular proteins, or functional assays, researchers can obtain comprehensive information about cell viability and the mechanisms by which it occurs in specific cell types or experimental conditions. The most commonly used dyes for viability detection
Dead cells: propidium iodide (PI) and 7-AAD, which bind to DNA but can only enter cells with damaged membranes, allowing dead cells to fluoresce.
Apoptotic cells: annexin V: a protein with a strong binding affinity for phosphatidylserine, exposed to the outer surface of the plasma membrane in the early stages of apoptosis, annexin V+PI is a commonly used combination to distinguish apoptotic cells from necrotic cells. Live cells: calcein AM, CFDA (carboxyfluorescein diacetate), FDA (fluorescein diacetate): enter living cells, but only fluoresce when interacting with intracellular enzymes Proliferation: CFSE (CFDA-SE) penetrates the cell membrane, covalently binds to intracellular proteins in living cells, and releases green fluorescence after hydrolysis. During cell division and proliferation, its fluorescence intensity decreases with cell division, and the labeled fluorescence can be evenly distributed among the two daughter cells, so its fluorescence intensity is half that of the parent cells, according to this characteristic, it can be used to detect cell proliferation, cell cycle estimation and cell division.
Front Immunol. 2013
Three. Cell cycle analysis
From the early days of flow cytometry, cell cycle analysis has been a valuable application.
The principle is based on the relationship between the amount of fluorescence and nucleic acids. Commonly used nucleic acid binding dyes: propidium iodide (PI), Hoechst, DAPI, 7-AAD, ethidium bromide, etc. Flow cytometry cell cycle fractions can have many applications, for example, DNA/Ki67 assays can combine phenotypic selection with cell cycle analysis for monitoring p53 cell cycle arrest, assessing anticancer activity, multidrug resistance, etc.
Four. Ion channel assays
Calcium plays a vital role in many cell signaling pathways as a key second messenger. It is particularly important in the activation of immune cells, including T cells, B cells, and NK cells.
T cell calcium ion signal (Nat Rev Immunol. 2019) In addition, calcium signaling is also involved in mast cell degranulation, neuronal excitability, synaptic transmission, and neurotransmitter release crucially.
Early measurements of cell degranulation were determined by flow cytometry using calcium ionophore A23187. Commonly used fluorescent dyes: fluo-3 and indo-1. While Ca2+ channel measurement is one of the most common applications, other ions such as magnesium, potassium, sodium, and hydrogen can also be monitored using flow cytometry.
Five. Cell function assays
The earliest assay was cellular esterase. The oxidation potential of granulocytes is detected using reactive dyes that respond to changes in oxidation states. For example, hydroethidine is used for neutrophil respiration bursts. Dichlorofluorescein diacetate, which has been used to study phagocytic cell function.
效应细胞杀伤功能，是流式细胞术的另外一个重要应用。 （参考文献F., Sunga, G.M., Arango-Saenz, D., Rossetti, M. A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity. J. Vis. Exp. (126), e56191, doi:10.3791/56191 (2017).
Cytokines are important executive molecules of immune cell function, which are crucial for scientific research, immune cell therapy, and clinical diagnosis and treatment. Multiplex cytokine assays developed based on flow cytometry have been widely used. Six. Protein engineering
Flow cytometry and sorting have not traditionally been one of the most commonly used techniques in protein engineering. However, in recent years, there have been more and more applications in this field.
Cells 2023 flow cytometry is used for enzymatic protein research, including cytochrome P450, glucose oxidase, chitinase, cellulase, peroxidase, esterase, transferase, β galactosidase, thiolactesterase, and others. Protein engineering, which involves introducing mutations (random or specific) at the gene level to create libraries consisting of thousands to millions of individual protein variants (as shown above), using flow cytometry Ability to analyze up to 10^8–10^9 clones per day and classify clones with desired characteristics. Seven. Sorting of mammalian and bacterial cells
Cell sorting is one of the important applications of flow cytometry, and mammalian cells are relatively mature and will not be repeated. The application of bacterial cells has also gradually begun to be established. In contrast to time-consuming traditional agar plating assays, flow sorting can quickly detect and sort individual bacterial cells in suspension. Despite the high performance of cell sorters, their use in microbiology has been limited. This is mainly due to the small size of microorganisms, which makes it difficult to distinguish them from cell debris or background particles in the medium. Another potential problem is that there are often no antibodies specific to bacterial strains. Other factors limiting the applicability of cell sorters for bacterial detection and sorting are primarily related to the sorter hardware capabilities themselves, and in the early days of flow cytometry instrumentation, the limited number of lasers and detectors limited the use of one or two fluorochromes at a time. With the development of the latest instruments, multi-laser and detection instruments have been developed: including Thermo Fisher's Bigfoot Spectral Cell Sorter, BD FACSAria III Sorter, Sony MA900 Cell Sorter, and Beckman Coulter's MoFlo Astrios EQ, among others. In addition, some pathogenic bacteria need to be performed in an experimental environment above BSL2, and some flow cytometry is now carried with BSL2 hood.
Cells 2023八.液滴微流体
Droplet microfluidics is a relatively new field focused on the formation, manipulation, and analysis of discrete droplets containing cells or DNA in picoliter volumes for applications in biology, chemistry, materials science, and medicine. In biology, droplet microfluidics enables single-cell analysis, high-throughput screening of biomolecules, cellular heterogeneity studies, and drug discovery. Flow cytometry analysis is a powerful technique for studying single cells, providing valuable information on a wide range of parameters. However, its measurement is limited to molecules directly attached to the cell, such as surface or intracellular markers, limiting the study of molecules secreted by cells or produced by DNA molecules but not physically attached. Droplet microfluidics offers a new way to overcome this limitation. Encapsulating cells or DNA in a single droplet creates discrete compartments that enable the analysis of compounds released or produced by encapsulated entities.
DE droplet analysis via sdDE-FACs. ( Lab Chip 2020)九.下一代生物制剂
Biopharmaceuticals already account for a significant share of the drug market, including therapeutic proteins (65%), vaccines (20%), and others. Sequencing (NGS) for single B cell bank analysis and clonal amplification identification, directly cloning immunoglobulin genes from human survivors, and isolating high-affinity neutralizing antibodies, have accelerated the development of monoclonal antibody drugs, however, this method is expensive and still requires follow-up functional validation. New strategies that combine single-cell isolation with functional screening using flow cytometry, MACS, or microfluidics to reduce development costs and eliminate failed drug candidates are the new application development directions for flow cytometry.
参考资料Crosland-Taylor, P.J. A device for counting small particles suspended in fluid through a tube. Nature 1953, 171, 37–38.Hamori, E.; Arndt-Jovin, D.J.; Grimwade, B.G.; Jovin, T.M. Selection of viable cells with known DNA content. Cytometry 1980, 1, 132–135.: Robinson, J.P.; Ostafe, R.; Iyengar, S.N.; Rajwa, B.; Fischer, R. Flow Cytometry: The Next Revolution. Cells 2023, 12, 1875. https://doi.org/10.3390/ cells12141875Gennady Bocharov et al, Asymmetry of Cell Division in CFSE-Based Lymphocyte Proliferation Analysis,Front Immunol. 2013 Sep 2;4:264. doi: 10.3389/fimmu.2013.00264Brower, K.K.; Carswell-Crumpton, C.; Klemm, S.; Cruz, B.; Kim, G.; Calhoun, S.G.K.; Nichols, L.; Fordyce, P.M. Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery. Lab Chip 2020, 20, 2062–2074.
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