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Flow cytometry
The principle of flow cytometry is to perform multiparametric, rapid quantitative analysis of individual cells or other biological particles at the molecular level using monoclonal antibodies. Flow cytometry is widely used in routine laboratory experiments. This article provides a brief introduction to the basic principles of flow cytometry and guidelines for selecting commonly used fluorescent dyes.
Flow cytometry (FCM) is a cutting-edge, high-tech instrument that integrates laser technology, electronic physics, photoelectric measurement, computer science, cellular fluorescence chemistry, and monoclonal antibody technology. In essence, flow cytometry is a technique for the multiparametric, rapid, quantitative analysis and sorting of cells or biological particles as they pass through a fast, laminar flow.
The structure of a flow cytometer is generally divided into three main components: the fluidics system, the optical system, and the signal detection and analysis system.
1. Liquid Flow System
The flow cytometry system is the core component of the instrument, where the sample to be analyzed intersects with the laser beam. The flow chamber is filled with sheath fluid, which serves to encase the sample stream in a sheath. This sheath flow is a stable, laminar flow that hydrodynamically focuses the sample stream, ensuring that the sample remains centered on the axis of the flow and that each cell spends an equal amount of time within the laser interrogation zone. As a result, accurate fluorescence measurements are obtained. Additionally, the sheath fluid helps prevent blockage of the nozzle while maintaining cell viability.
2. Optical System
The optical system primarily comprises a laser and filters. Currently, most benchtop flow cytometers employ argon-ion gas lasers. Lasers are coherent light sources that provide monochromatic, high-intensity, and highly stable illumination, making them ideal for the rapid analysis of weak cellular fluorescence. The intensity of the fluorescent signal emitted by the fluorophores carried by each cell is dependent on both the duration of illumination and the intensity of the excitation light; therefore, the cells must be exposed to sufficient light intensity.
The commonly used lasers include:
Violet laser with a wavelength of 405 nm
Blue laser with a wavelength of 488 nm
Green laser with a wavelength of 561 nm
Red laser with a wavelength of 633 nm
Optical filters are primarily classified into three types: long-pass filters (LP), short-pass filters (SP), and band-pass filters (BP).
(1) Long-pass filter: A long-pass filter allows light with wavelengths longer than a specified cutoff to pass through while blocking or attenuating light with wavelengths shorter than that cutoff. For example, an LP500 filter permits wavelengths greater than 500 nm to pass while absorbing or reflecting wavelengths at or below 500 nm.
(2) Short-pass filter: Contrary to a long-pass filter, this type transmits light below a specified wavelength while absorbing or reflecting light at and above that wavelength. For example, an SP500 filter allows light with wavelengths below 500 nm to pass through, while blocking or reflecting light at and above 500 nm.
(3) Bandpass filters: Bandpass filters allow light within a relatively narrow wavelength range to pass through. Typically, such filters are labeled with two numbers: one indicating the center wavelength of the passband, and the other specifying the width of the passband. For example, BP500/50 denotes a passband ranging from 475 nm to 525 nm.
3. Signal Detection and Analysis System
When cells are labeled with a fluorescent probe and exposed to a laser beam, they are excited by the laser, generating signals that represent various intracellular components,
Fluorescent signals at different wavelengths are emitted in a 360-degree spatial solid angle centered on the cell, generating both scattered light and fluorescent signals. The electronic system converts these analog signals into digital form, calculates the height, width, and area of each pulse peak, and transmits the data to a connected computer.
(1) Scattered light signals: Scattered light is divided into forward scatter (FSC) and side scatter (SSC). Since scattered light does not depend on any cell-sample preparation techniques, such as staining, it is referred to as a physical parameter of the cell, or an intrinsic parameter.
① Forward-angle scattering: Forward-angle scattering is related to the size of the cells being measured, specifically to the square of the cell diameter. In FCM applications, FSC is typically used as a threshold to exclude debris and small particles in the sheath fluid, thereby minimizing interference with the cells under analysis.
② Lateral angle scattering: Lateral angle scattering refers to the scattered light signal at a 90° angle relative to the laser beam. This type of scattered light is more sensitive to the refractive indices of the cell membrane, cytoplasm, and nuclear membrane, thereby providing information on the fine intracellular structures and particle characteristics.
Figure 1: Scatter plots from a flow cytometer
(2) Fluorescence signals: When the laser beam is oriented perpendicular to the cell, two types of fluorescence signals are typically generated. One is the weak intrinsic fluorescence emitted by the cell itself upon laser irradiation, known as cellular autofluorescence; the other is the fluorescence signal produced after the cells have been labeled with specific fluorophores and subsequently excited by the laser. By detecting and quantitatively analyzing these fluorescence signals, it is possible to determine the presence and quantify the relevant cellular parameters under investigation.
Figure 2: Principle of Laser Operation
Figure 3: Workflow for Flow Cytometry
Fresh solid tissue samples can be processed using mechanical disruption, enzymatic digestion, or chemical treatment. Peripheral blood samples are typically processed by density-gradient centrifugation, isolation of peripheral blood mononuclear cells (PBMCs), or lysis of red blood cells followed by recovery of leukocytes. Different brands of lysis buffers may require different lysis conditions; therefore, it is recommended to follow the protocol provided in the manufacturer’s instructions.
Typically, post-lysis peripheral blood samples are clearly divided into three populations: the granulocyte population, the monocyte population, and the lymphocyte population. In contrast, fresh apheresis samples contain very few granulocytes and are predominantly composed of two populations: the lymphocyte population and the monocyte population.
Figure 4: In-machine performance after fresh single-donor red cell separation Figure 5: Instrument performance after whole-blood hemolysis
Note that when our next experiments involve intracellular or nuclear staining, we should, whenever possible, use isolated PBMCs for flow cytometry to minimize the impact of red cell lysis on cell viability.
Figure 4: Procedure for Single-Cell Staining
When staining single-cell suspensions, careful consideration should be given to the choice of fluorophore. The following principles can be followed:
1. Select fluorochromes based on the instrument configuration: Principles for multicolor labeling: only one fluorochrome may be used per channel; however, fluorochromes from different channels can be freely combined. Common four-color combinations include FITC, PE, PerCP, and APC.
2. Allocate fluorophores appropriately based on the strength of antigen expression: First-tier antigens: These antigens are typically stable in expression and often exhibit high levels of expression. When such antigens are highly expressed, they can be paired with antibodies conjugated to lower-intensity fluorophores, such as Alexa Fluor 700, Brilliant Violet 570™, Brilliant Violet 785™, or APC-Cy7. Second-tier antigens: These antigens are critical for further phenotypic characterization; however, their expression levels can vary across parallel experiments. Due to their variable expression, they are generally best paired with antibodies conjugated to medium-intensity fluorophores, such as Brilliant Violet 510™, Brilliant Violet 650™, FITC, or PerCP-Cy5.5. Third-tier antigens: The expression of these antigens may differ substantially among samples, or their expression levels may even be unknown. Typically, only purified antibodies targeting these antigens are commercially available, and the range of compatible fluorophores is limited. For detection of such antigens, it is advisable to select antibodies conjugated to the highest-intensity fluorophores, such as Brilliant Violet 421™, PE, APC, or PE-Cy7.
3. Select fluorophores with minimal spectral overlap: Whenever possible, choose fluorophores with limited spectral overlap, such as FITC/PE-Cy7; alternatively, select fluorophores excited by different lasers, such as FITC/APC and PE/APC.
4. Minimize false positives caused by the use of conjugated dyes. Conjugated fluorescent dyes refer to two fluorophores linked together to enable fluorescence resonance energy transfer. When using conjugated fluorophores, it is essential to complete the experiment as quickly as possible; the longer the exposure time, the more unstable the fluorophores become.
The fluorescence intensity of staining is correlated with the staining index: the higher the staining index, the stronger the fluorescence; the lower the staining index, the weaker the fluorescence. Ranked by staining index from strongest to weakest, the order is PE > APC > FITC > PerCP.
References: Wang Shu-kui, “Using Flow Cytometry Color Schemes”
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