Flow cytometry is a fluorescence-based assay that simultaneously assays multiple properties, such as cell population count and protein abundance, from a single cell suspended in solution. It is a powerful tool that enables rapid, quantitative and accurate determination of cell properties and provides superior interpretation of cell population heterogeneity.
This is done using instruments that guide individual cells through a light source and measure the scattering and emission of light energy produced at various wavelengths. The researchers used fluorescent molecules with different excitation and emission properties to be able to merge, detect, and distinguish them to obtain multiple readings in a single cell. These fluorescent molecules can be attached to antibodies that target specific proteins or protein modifications, or they can be used as dyes to bind directly to cellular components.
The basic principle is to arrange the cells in the sample in a single row through fluid mechanics, shine a laser light and detect the light scattering and fluorescence signals of individual cells.
First, the sample to be tested (such as blood, cell suspension) forms a single-deficiency flow through the sheath flow. In the flow process, the cells successively pass through the point of exposure of the laser beam. The laser interacts with the cell to produce both forward scattered light (FSC) and lateral scattered light (SSC). The FSC provides information about the cell's volume, while the SSC reflects the complexity of the cell's internal structure. In addition, cells can be pre-labeled with specific fluorescent dyes or antibodies that fluoresce at specific wavelengths when excited by a laser. Flow cytometry's photodetectors record scattered light and fluorescence signals, convert them into electronic signals, and analyze them through a computer. Through a combination of multiple laser and fluorescent dyes, flow cytometry enables simultaneous analysis of multiple parameters, such as cell size, shape, surface marker expression, nucleic acid content.
A flow cytometer consists of a fluidics system for cell processing and an optical system that includes a data acquisition signal detector and processor. The fluidics system controls the movement of the cell suspension, ensuring that cells are aligned in a single file before they pass through the laser. The optics system includes lasers and a series of mirrors and filters that direct the laser light onto the cells and capture the scattered and emitted light from the cells. The electronic system is responsible for processing the light signals, converting them into digital data that can be visualized and interpreted by the user.
Bioconjugation refers to the chemical linking of two biomolecules, often involving a recognition molecule (e.g., antibody) and a signaling molecule (e.g., fluorochrome). In flow cytometry, bioconjugation is used to tag cells with fluorescent markers, enabling the detection of specific antigens on or within cells.
Bioconjugation significantly enhances both detection sensitivity and specificity in flow cytometry. Fluorescent tags, when conjugated to antibodies or ligands, provide a measurable signal upon binding to their corresponding antigens. By using multiple fluorophores with distinct emission spectra, researchers can simultaneously track different biological markers, creating a high-dimensional dataset from a single sample. This multiplexing capability is a major advantage, allowing for the detailed characterization of complex cellular environments, such as immune cell subtypes or tumor microenvironments.
Additionally, bioconjugation increases the accuracy of flow cytometric data by ensuring that the detection reagents bind specifically to their intended targets. This reduces background noise and non-specific interactions, improving the reliability of the data and enabling more precise quantification.
The following are some common bioconjugates utilized in flow cytometry.
Antibody-drug conjugates (ADCs): These bioconjugates consist of antibodies linked to cytotoxic drugs. ADCs are designed to selectively deliver drugs to cancer cells while minimizing effects on healthy tissues. In flow cytometry, they are used to quantify target antigens on cell surfaces, providing insight into tumor characteristics and aiding in therapeutic decision-making.
Fluorescently labeled antibodies: Common fluorochromes include fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC). These labeled antibodies enable the detection and quantification of specific proteins on the surface of cells, allowing researchers to analyze cellular phenotypes and activation states.
Peptide-MHC complexes: These bioconjugates consist of peptide antigens bound to major histocompatibility complex (MHC) molecules. They are critical for studying T-cell responses in immunology. Flow cytometry enables the assessment of T-cell receptor recognition and specificity by using these complexes to stain T cells, providing valuable data on immune responses.
Enzyme-linked probes: Bioconjugates that consist of enzymes linked to detection agents can be used for signal amplification in flow cytometry. For instance, horseradish peroxidase (HRP) or alkaline phosphatase can be conjugated to antibodies, facilitating enhanced signal detection.
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Covalent bonding methods: Covalent bonding is a reliable and stable biological binding method, which binds biomolecules and detectable labels by forming permanent chemical bonds to ensure that the combination does not separate during the experiment, and is suitable for long-term preservation and repeated detection. Common methods include crosslinkers (e.g. EDC, NHS), which form a stable link between the amino acid and the active group of the label and are suitable for situations that do not affect the biological activity of the antibody or ligand. Click chemistry utilizes biological orthogonal reactions (such as azide-acetylene cycloaddition) to achieve high specificity and efficient binding under mild conditions. The maleimide-mercaptan reaction is often used to bind molecules containing mercaptan groups to maleimide activation tags suitable for labeling proteins or peptides..
Non-covalent bonding methods: Non-covalent binding methods utilize the natural affinity between biomolecules rather than permanent chemical bonds. Although less stable than covalent methods, non-covalent methods offer flexibility, allowing reversible binding, to be used when permanent binding is not required or reversible interactions provide experimental advantages.
The biotin-avidin system is one of the most commonly used non-covalent binding methods. Biotin binds antibodies or proteins with ease, while avidin or streptavidin binds biotin with very high affinity, and biotinylated antibodies are detected by avidin fluorescent probes for highly sensitive and reversible assays in flow cytometry, suitable for multiple assays. Hydrophobic interactions use hydrophobic regions in proteins to bind hydrophobic dyes or fluorescent molecules non-covalently. Electrostatic interactions, which bind biomolecules with opposite charges, are suitable for labeling charged fluorescent dyes, but are sensitive to pH and ionic strength.
One of the most prominent applications of bioconjugation in flow cytometry is immunophenotyping, a process that enables the identification and quantification of different cell types within a heterogeneous sample based on their surface proteins. Through bioconjugation, antibodies specific to these proteins are labeled with fluorescent dyes, which allows for the simultaneous detection of multiple cell types. For example, T cells, B cells, and monocytes can be differentiated based on the expression of unique surface markers, with each marker conjugated to a distinct fluorochrome. The multi-parametric nature of this analysis allows researchers to gain a deeper understanding of the immune system's composition and dynamics, which is crucial for both basic research and clinical studies in immunology.
Another significant application of bioconjugation in flow cytometry is in cell sorting, particularly fluorescence-activated cell sorting (FACS). In this technique, cells labeled with fluorescently conjugated antibodies are sorted based on their fluorescence signal, allowing researchers to isolate specific subpopulations of interest. For example, FACS can be used to separate immune cells, stem cells, or cancer cells from a mixed population for further study or experimental manipulation. The accuracy and speed of cell sorting are greatly enhanced by the use of highly specific bioconjugates, enabling the isolation of rare cell populations with minimal contamination. Moreover, bioconjugation allows for sorting based on functional markers, such as phosphorylated proteins, providing insights into intracellular signaling pathways and cellular responses to stimuli.
In cancer research, flow cytometry can be used to detect biomarkers on the surface of tumor cells, such as HER2 or CD20, through the use of fluorescently labeled antibodies. This allows for the rapid quantification of biomarker expression levels, which can inform treatment decisions and predict patient outcomes. Additionally, intracellular biomarkers, such as phosphorylated proteins involved in signal transduction pathways, can be detected using bioconjugated antibodies in a technique known as phospho-flow cytometry. This application provides insights into cellular processes such as proliferation, apoptosis, and immune activation, making it a powerful tool for both basic research and therapeutic development.
The combination of bioconjugation technology with mass spectrometry CyTOF and other cutting-edge technologies has significantly expanded the application range of flow cytometry. By using metal-labeled antibodies instead of traditional fluorescent dyes, CyTOF was able to simultaneously detect more than 40 characteristic parameters at the single cell level. Bioconjugating ensures the strict binding of metal isotopes to specific antibodies, thus providing a new approach for high-dimensional cell studies in immunology, oncology and systems biology.
With the application of artificial intelligence (AI) and machine learning (ML) techniques in the field of flow cell data analysis, researchers are able to efficiently process large amounts of complex information generated by high-dimensional experiments. The fusion of AI and bioconjugated technology not only improves the accuracy of cell subsets identification, but also promotes a deeper understanding of disease mechanisms and accelerates the screening process of potential therapeutic targets.
The application of bioconjugation in flow cytometry is promising. As new technologies and conjugates continue to emerge, flow cytometry will remain an indispensable tool in basic and applied biology research. These advances not only increase the precision and depth of cell analysis, but also drive innovation in areas such as personalized medicine, immunotherapy, and drug discovery. As bioconjugation technology evolves, its integration with other cutting-edge platforms will further push the boundaries of what is possible, providing unparalleled insights into the complexity of biological systems.