Flow Cytometry: Definition, Principles, Protocols, Dyes, and Uses

Flow cytometry (FCM) is a technique for rapidly analyzing cell characteristics in a liquid flow. It enables multi-parameter quantitative analysis of single cells by combining them with specific antibodies, measuring attributes such as cell surface markers, intracellular protein expression, cell cycle, and DNA content. Flow cytometry offers advantages such as high throughput, high sensitivity, and high specificity, making it widely applicable in fields like immunology, hematology, and oncology.

What is Flow Cytometry?

Flow cytometry is a fluorescence-based detection method that simultaneously measures multiple characteristics of single cells suspended in a solution, such as cell population counts and protein abundance. It is a powerful tool for the rapid, quantitative, and accurate assessment of cellular characteristics and provides excellent insights into cell population heterogeneity. This process involves directing single cells to flow through a light source, where light scattering and emissions at various wavelengths are measured using an instrument. Researchers use fluorescent molecules with distinct excitation and emission properties to combine, detect, and distinguish signals, enabling multiple readouts from single cells. These fluorescent molecules can be attached to antibodies targeting specific proteins or protein modifications, or they can directly bind to cellular components as dyes. The key advantages of flow cytometry include:

Fig. 1. Flow cytometry principle.

• High Throughput: Analyzes thousands to millions of cells per experiment, enabling large-scale data collection within seconds.
• Single-Cell Multi-Parameter Analysis: Simultaneously measures multiple parameters of single cells, such as cell size, granularity, surface markers, and internal complexity.
• High Data Accuracy: Combines fluorescence signals with physical parameters like cell size, providing precise subpopulation analysis.

How Does Flow Cytometry Work?

In a flow cytometer, cells are encapsulated in a sheath fluid and ejected through a nozzle at a specific speed. As cells pass through the laser beam one by one, the laser illuminates the cells and generates light signals, which are collected by detectors, converted into electrical signals, and transmitted to a computer for further processing and analysis. Since the number of target antigens varies among cells, the intensity of the light signals also differs, enabling multiparametric quantitative analysis of each cell. The light signals include scattered light signals and fluorescent signals. Scattered light is categorized into forward scatter (FSC) and side scatter (SSC), with intensities determined by the physical properties of the cells, such as cell size and internal structural complexity. Fluorescent signals are generated when the target antigens bind to fluorescently labeled antibodies and are excited by the laser beam.

Flow Cytometry Protocol

The basic operation process of flow cytometry mainly includes:

1. Sample Preparation: Samples can originate from tissues, blood, or other liquids, all of which must ultimately be prepared into single-cell suspensions to facilitate staining and detection.
2. Cell Staining: Flow cytometry requires staining cells to mark specific antigens. Staining methods are broadly divided into two categories: surface antigen staining and intracellular antigen staining. For surface antigen staining, cells are directly incubated with fluorescently conjugated antibodies. For intracellular antigen staining, cells must undergo fixation and permeabilization before incubation with fluorescently conjugated antibodies.
3. Cell Washing: During staining, washing the cells with a wash buffer is necessary to remove unbound antibodies and other impurities. The wash buffer typically consists of pre-cooled PBS with the optional addition of 2% BSA or 2% FBS to improve cell viability during handling. Washing reduces non-specific binding and background interference, enhancing the specificity and accuracy of the experiment.
4. Cell Fixation: For intracellular antigen detection, fixation reagents (e.g., 4% paraformaldehyde) and permeabilization agents (e.g., 0.1% Triton X-100) are used to fix and permeabilize cells. Fixatives reduce cellular activity and deformation, preserving cell morphology and antigen location stability. Permeabilization agents facilitate the entry of antibodies to bind intracellular antigens.
5. Data Acquisition: During data acquisition, cells pass through lasers that excite the markers, generating fluorescence signals that are captured by detectors. The collected data is typically saved in Flow Cytometry Standard (FCS) files. FCS files contain multi-parameter values for each cell, such as fluorescence intensity, enabling researchers to analyze individual cell characteristics and their distributions.

Flow Cytometry Staining Protocol

The flow cytometry staining protocol is a critical step for analyzing cell phenotypes, functions, and underlying mechanisms. By using fluorescently labeled antibodies, flow cytometry can precisely identify specific proteins on the cell surface or within cells, revealing developmental stages, immune phenotypes, and molecular functions. Common staining protocols include surface protein staining and intracellular protein staining, used to analyze immune characteristics of cell populations and internal molecular expressions, respectively. Optimizing the staining process and reaction conditions is essential for obtaining high-quality flow cytometry data.

• Cell Surface Protein Staining

Using fluorescently conjugated antibodies to label cell surface markers followed by flow cytometry analysis allows identification of cell subsets and their functions based on lineage and developmental stages. These surface markers include receptors for soluble and cell-bound sites, ion channels, glycoproteins, phospholipids, and more. For example, CD4 is a surface marker for helper T cells. Further differentiation of cell subsets can be achieved based on chemokine receptors and cluster of differentiation (CD) antigens. Live cells stained with antibodies can be sorted according to their unique phenotypes for use in other experiments. The cell surface staining workflow includes:

• Prepare single-cell suspensions.
• [Optional] Block Fc receptors (10-20 minutes).
• Add antibodies and incubate at 2-8 °C (30 minutes).
• Wash cells with flow cytometry staining buffer (1-2 x 5 minutes).
• If using unconjugated primary antibodies, add secondary reagents and incubate at 2-8 °C (30 minutes).
• Wash cells with flow cytometry staining buffer (1-2 x 5 minutes).
• [Optional] Stain cells with viability dyes (20-30 minutes).
• [Optional] Lyse red blood cells using 1X RBC lysis buffer or 1X fix/lyse buffer (10-12 minutes).
• Resuspend cells in flow cytometry staining buffer.
• Analyze cells using a flow cytometer.

• Intracellular Protein Staining

Protein transport inhibitors can block the secretion of proteins such as cytokines or chemokines, retaining them within the cell, which can then be analyzed by flow cytometry. Common inhibitors include the endoplasmic reticulum transport inhibitor Brefeldin A and the Golgi transport inhibitor Monensin, which can be used alone or in combination with other stimulants. Different cytokines and chemokines may respond differently to various inhibitors. Cells exposed to inhibitors for prolonged periods may undergo cell death, so conditions must be optimized based on the target protein and cells being analyzed. Fixation is the process of preserving or stabilizing biological material, and the fixation time and strength must be optimized to preserve antigens and cell structure while minimizing antigen epitope damage. If the target antigen is an intracellular protein, permeabilization is required to allow antibodies and reagents to enter the cell and bind to the target protein. Fixation and permeabilization conditions should be optimized based on the intracellular location of the target protein. The staining of cell surface proteins after fixation depends on how the fixation process affects protein epitopes. The staining workflow for intracellular proteins primarily includes:

• Prepare single-cell suspensions.
• [Optional] Block Fc receptors (10-20 minutes).
• Add surface protein antibodies and incubate at 2-8 °C (30 minutes).
• Wash cells with flow cytometry staining buffer (1-2 x 5 minutes).
• [Optional] Stain cells with viability dyes (20-30 minutes).
• [Optional] Lyse red blood cells using 1X RBC lysis buffer or 1X fix/lyse buffer (10-12 minutes).
• Fix and permeabilize cells (30-90 minutes).
• Wash cells with 1X permeabilization buffer (1 x 5 minutes).
• Add antibodies for intracellular proteins and incubate at 2-8 °C (30 minutes).
• Wash cells with 1X permeabilization buffer (1-2 x 5 minutes).
• Resuspend cells in flow cytometry staining buffer.
• Analyze cells using a flow cytometer.

Fluorescent Dyes for Flow Cytometry

There is a wide variety of fluorescent dyes available, each with unique molecular structures resulting in distinct excitation and emission spectra. When selecting a dye or a fluorophore-conjugated monoclonal antibody, the laser wavelength of the instrument's configuration must be considered, ensuring that the laser's excitation wavelength aligns closely with the dye's excitation spectrum peak. Additionally, the dye's emission spectrum (fluorescence color) must match the detection channel of the instrument. Over years of development, numerous fluorescent dyes, including monomeric dyes, tandem dyes, and fluorescent proteins, are now available on the market, with new dyes continuously emerging.

• Monomeric Dyes

Traditional dyes such as FITC, PE, and APC have been widely used for years, while new monomeric dyes like Bio-Rad's StarBright series are gradually being introduced. These newer dyes offer superior photostability and brighter fluorescence compared to traditional dyes. Optimized monomeric dyes enable better compatibility with multicolor experimental designs and multicolor flow cytometers, greatly reducing the complexity of dye selection in multicolor flow cytometry.

• Tandem Dyes

Unlike monomeric dyes, tandem dyes are created by covalently coupling one monomeric dye to another, using Förster Resonance Energy Transfer (FRET) for excitation. Examples include PE-Cy5, PerCP-Cy5.5, and APC-Cy7. Tandem dyes simplify laser configuration requirements, expanding multicolor flow cytometry capabilities. However, tandem dyes depend on FRET efficiency, which can vary between manufacturers or batches. Additionally, tandem dyes have lower stability, and long-term storage may cause the covalent bond between the two dyes to break. These factors can affect fluorescence compensation or spectral analysis, so special attention is required during use.

• Fluorescent Proteins

Common fluorescent proteins include GFP, YFP, mCherry, and tdTomato. These have become indispensable tools in scientific research for monitoring protein expression, typically through co-expression or fusion with target proteins. In flow cytometry experiments, fluorescent proteins allow the quantification of intracellular markers in live cells without the need for membrane permeabilization.

What is Flow Cytometry Used For?

Flow cytometry enables the simultaneous acquisition of multiple parameters about cell surface and intracellular characteristics, including cell size, morphology, molecular marker expression, and functional states. Its core applications in immunology and cell biology include immunophenotyping, cell cycle analysis, apoptosis detection, and cell viability assessment.

• Immunophenotyping

Immunophenotyping is a major application of flow cytometry, widely used for identifying and quantifying different immune cell subsets. By using fluorescently labeled specific antibodies, flow cytometry can detect characteristic molecules on the cell surface (e.g., receptors, antigens, and cell markers), enabling the differentiation of immune cells such as T cells, B cells, dendritic cells, monocytes, and natural killer (NK) cells. Immunophenotyping not only reveals the composition of immune cell subsets but also reflects their functional states, helping researchers understand immune responses. For example, in cancer immunomonitoring, flow cytometry is utilized to detect changes in immune cells within the tumor microenvironment and to study immune evasion mechanisms.

• Cell Cycle Analysis

Cell cycle analysis is another critical application of flow cytometry, particularly for evaluating the distribution of cell populations across different cell cycle phases. By staining cellular DNA (e.g., using PI or DAPI dyes), flow cytometry accurately measures the DNA content within cells to determine their cell cycle phases—such as G0/G1, S, and G2/M phases. This analysis is essential for cancer cell proliferation studies, mechanisms of drug action, and cell growth regulation research. Additionally, cell cycle analysis can be applied to screen anticancer drugs and investigate the effects of cell cycle inhibitors or promoters.

• Apoptosis Detection

Apoptosis detection is a key flow cytometry application in cell death research. By utilizing specific fluorescent dyes such as Annexin V and PI (propidium iodide), flow cytometry can distinguish early apoptosis, late apoptosis, and necrotic cells. Annexin V binds to exposed phosphatidylserine (PS), an early marker of apoptosis, while PI stains cells that have lost membrane integrity, identifying late apoptotic or necrotic cells. Apoptosis detection is widely used in drug screening, cancer therapy research, and immune response analysis, helping researchers understand cell death mechanisms and evaluate treatment efficacy.

• Cell Viability Assessment

Cell viability assessment uses live and dead cell dyes (e.g., Calcein-AM and PI) to evaluate the survival status of cells. Live cell dyes penetrate intact cell membranes and are hydrolyzed by intracellular enzymes to produce fluorescent signals, while dead cell dyes enter cells with damaged membranes and emit fluorescence. Flow cytometry rapidly provides viability data for different cell populations, assessing the impact of drugs, chemicals, or environmental factors on cell viability. This application plays a vital role in toxicology research, drug screening, cell therapy, and fundamental life science studies.

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