Flow Cytometry (FCM/FC) Protocol and FAQs

Flow cytometry is a new technology developed in the late 1960 s, which uses flow cytometer to quickly and quantitatively analyze the physical and chemical characteristics of cell population and accurately sort cells according to these physical and chemical characteristics. It mainly includes two parts : flow analysis and flow sorting. Flow cytometry reflects the physical and chemical characteristics of cells by receiving scattered light signals and fluorescence signals after laser irradiation of cells in the flow, such as cell size, particle size and expression of antigen molecules.

The Principle of Flow Cytometry

The laser beam of a specific wavelength directly illuminates the cells in the high-pressure-driven fluid flow, and the generated optical signal is received by multiple receivers. One is the scattered light signal (forward angle scattering) received in the linear direction of the laser beam, and the other is the light signal received in the vertical direction of the laser beam, including the scattered light signal (lateral angle scattering) and the fluorescence signal. Cells or particles with a diameter of 0.2 ~ 150 μm suspended in the liquid flow can scatter the laser beam, and the fluorescein bound on the cells can emit fluorescence after being excited by the laser. The scattered light signal and the fluorescence signal are received by the corresponding receiver, and the physical and chemical characteristics of each cell can be reflected according to the fluctuation of the received signal.

Flow cytometry has three major elements, respectively, flow cytometry, sample cells and fluorescent dyes or fluorescein-conjugated antibodies.

The object of flow cytometry was single cell suspension. Flow cytometry cannot directly detect cells in tissue blocks. To detect cells in organs or tissues, organs or tissues must be prepared into single cell suspensions by various methods, and then labeled with fluorescein-conjugated antibodies before they can be detected by flow cytometry. Flow cytometry cannot directly detect molecules, but it can indirectly detect molecules by using synthetic particles instead of cells and then combining the antibody of the molecule with artificial particles, such as CBA method to detect cytokines.

The Steps of Flow Cytometry

Sample Preparation

Sample preparation is one of the most critical steps in flow cytometry experiments. Ensuring the quality of cell suspension directly affects the accuracy of experimental results. The specific steps are as follows :

Cell separation

Cells were extracted from tissues or blood, which may be preliminarily isolated by centrifugation, cell screening and other methods.

Cell Counting

The appropriate cell concentration (generally 1 × 10 to 1 × 10 cells / ml) is ensured by microscopic counting or automated cell counting instruments. Cell cleaning : Sometimes cells need to be cleaned to remove possible impurities and proteins in the solution, reducing interference with the analysis results. Cells are washed with PBS buffer.

Cell fixation (optional)

If the internal components of the cell need to be analyzed, the cell may need to be fixed. Commonly used fixatives such as formaldehyde, ethanol, etc.

Labeling cells

Labeling cells with fluorescent antibodies or other fluorescent dyes to detect specific molecules on the cell surface or in the cell during subsequent analysis.

Sample Injection

The treated cell suspension was introduced into the sample tube of the flow cytometer. The cell suspension is fed into a flow cytometer through a very fine tube (usually a micron-sized tube). Cells are guided to a single cell laminar flow state during flow.

Laser Irradiation

When the cells flow through the flow cytometer, the laser beam will irradiate each cell. The wavelength of the laser is selected according to the experimental requirements and the characteristics of the fluorescent marker. The commonly used laser wavelengths are 488 nm, 633 nm, etc.

Fluorescence labeling excitation

The fluorescent markers on the surface or inside of the cell will be excited by the laser and emit corresponding fluorescence. Each fluorescent marker emits different fluorescence signals at a specific wavelength.

Scattering light

In addition to fluorescence, the scattering of laser by cells is also an important signal for flow cytometry analysis. Scattering light can provide information about the physical properties of cells, especially the size and particle size of cells.

Optical Signal Detection

After laser irradiation, cells produce a variety of optical signals, including scattered light and fluorescent light signals. The flow cytometer is equipped with multiple optical detectors and filters to analyze these signals.

Forward scattering light (FSC)

The intensity of forward scattering light is related to the size (volume) of the cell. The larger the cell, the stronger the intensity of forward scattering light. Forward scattered light is usually used to measure the size of cells.

Lateral scattered light (SSC)

The intensity of lateral scattered light is related to the particle size of the cell (such as the structural complexity within the cell). Cells with higher particle size (such as white blood cells) produce stronger lateral scattering light. Lateral scattered light is used to analyze the internal structure of cells.

Fluorescence signal

The flow cytometer is equipped with multiple fluorescence detectors, each of which is responsible for capturing fluorescence signals in different wavelength ranges. Each fluorescent marker has a specific emission wavelength, and these signals are captured and analyzed by a suitable filter and detector.

Signal Processing And Data Analysis

The detected scattered light and fluorescence signals are converted into digital signals by flow cytometry, and processed and analyzed by computer. Data processing is usually divided into the following steps :

Signal conversion

All optical signals (scattered light and fluorescent light) are converted into electrical signals, amplified by an electronic amplifier, and then converted into digital signals for processing.

Data acquisition

Computer software receives the data of each cell and stores it. The forward scattering light, lateral scattering light and fluorescence signals of each cell were recorded to form a complete set of multidimensional data.

Data analysis

According to the purpose of the experiment, software can be used for various analysis. For example, specific molecules in cells can be quantified by fluorescence intensity, or different types of cell populations can be distinguished by scattered light intensity.

Results Interpretation And Visualization

The data generated by flow cytometry are usually displayed through charts (such as histograms, scatter plots, density maps, etc.). Common analysis results include :

Distribution of cell populations

The distribution of different cell populations on forward scattering and lateral scattering light was analyzed by scatter plot. Different types of cells (such as lymphocytes, monocytes, etc.) show different scattering patterns.

Detection of fluorescent dyes

The expression of specific molecules on the cell surface or inside was analyzed by the distribution of fluorescence signals. For example, the use of anti-CD-labeled antibodies can map the fluorescence intensity map of a specific immune cell population.

Cell cycle analysis

Cell cycle status (such as G0 / G1 phase, S phase, G2 / M phase, etc.) was analyzed by fluorescence signals of DNA dyes (such as PI, DAPI).

Figure 1. Schematic comparison of a conventional and spectral detection in flow cytometry. (Reference source: Spectral Flow Cytometry.）

Application of Flow Cytometry

Flow cytometry is widely used in life sciences, medicine, drug research and other fields. Common applications include :

Immunophenotypic Analysis

By labeling different antigens, it helps to determine the immune characteristics of the cell population.

Cell Cycle Analysis

Helps study cell proliferation and cell cycle status by labeling DNA.

Cell Viability Analysis

The physiological processes such as cell viability, apoptosis and autophagy were evaluated.

Cell Sorting

Accurate sorting based on the different characteristics of cells for further research.

Alpha Lifetech has many years of project experience. With its deep technical background and professional team, it can provide customized services to customers. Alpha Lifetech provides Flow Cytometry (FCM / FC) services to meet customers ' scientific research and clinical needs.

FAQ

1. Cell aggregation leads to the formation of overlapping signals of multiple cells in the instrument, which affects the accuracy of the data.

Cause : cell concentration is too high or too low. The cells were damaged or adhered during the treatment. The sample may contain impurities or undispersed cell clusters. Solutions : (1) Appropriate dilution of cells : ensure that the cell concentration is within the recommended range of the instrument, usually from 1 × 10 Ω to 1 × 10 Ω cells / mL. (2) Use of deaggregating agents : an appropriate amount of deaggregating agents, such as DNase, EDTA, etc., can be added to help disperse cells. (3) Optimize sample handling : Use sterile operation to avoid excessive oscillation or use too strong centrifugal force. (4) Accelerated filtration : Cell sieves (e.g., 70 μm or 40 μm aperture sieves) are used to filter samples before flow cytometry to remove large cell clusters and impurities.
2. Fluorescently labeled antibodies or dyes may cause uneven staining and affect the reliability of the data, especially in multi-label experiments.

Cause : improper dyeing steps or too short time. The concentration of dye is too high or too low. Damage or poor permeability during cell processing. Solutions : (1) Optimization of dyeing conditions : Adjust the concentration and time of dyeing according to the reagent manual, and usually ensure that the dyeing reaction is within the appropriate temperature and time range. (2) Use appropriate washing steps : After dyeing, remove unbound dyes or antibodies by adequate washing. (3) Use appropriate dyes : Select dyes and antibodies that match the experimental purpose to ensure their specificity and sensitivity. Pre-experiment : Before the formal experiment, a small-scale pre-experiment was carried out to optimize the dyeing conditions and dye concentration.
3. High background noise.

Cause : Non-specific binding of fluorescent dyes. There are non-target cells or particles in the sample. Improper setting of laser and detector. High background noise will affect the accuracy of the experimental results, making it difficult to distinguish the target signal from the background signal. Solutions : (1) Adjust the concentration of fluorescent dyes : avoid using excessive fluorescent dyes and reduce non-specific binding. (2) Optimize antibody selection : Use high-affinity antibodies to reduce background signals. (3) Use control samples : Evaluate background signals by negative, negative, or isotype controls to help distinguish between specific signals and background noise. (4) Calibration of laser and filter : Ensure that the instrument 's laser and filter settings are correct and minimize the impact of background noise.
4. Antibody selection and cross reaction: In multiple labeling experiments, cross-reactions between different antibodies may occur, resulting in difficulty in data interpretation.

Cause : Cross-reactivity between antibodies selected improperly or fluorescently labeled antibodies. The emission spectra of dyes overlap. Solutions : (1) Select high-specific antibodies : Use verified, high-affinity antibodies to ensure their specificity. (2) Avoid dye overlap : Select fluorescent dyes with different emission spectra to avoid signal overlap between different dyes. (3) Using the appropriate labeling sequence : The order of antibody labeling was reasonably arranged according to the order of excitation and emission spectra of fluorescent dyes.

reference

[1] McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. Published 2018 Feb 21. doi:10.1002/cpim.40

[2] Givan AL. Flow cytometry: an introduction. Methods Mol Biol. 2011;699:1-29. doi:10.1007/978-1-61737-950-5_1

[3] Nolan JP, Condello D. Spectral flow cytometry. Curr Protoc Cytom. 2013;Chapter 1:1.27.1-1.27.13. doi:10.1002/0471142956.cy0127s63

[4] Maciorowski Z, Chattopadhyay PK, Jain P. Basic Multicolor Flow Cytometry. Curr Protoc Immunol. 2017;117:5.4.1-5.4.38. Published 2017 Apr 3. doi:10.1002/cpim.26

[5] Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol. 2001;38(2):179-193. doi:10.1016/s0037-1963(01)90051-4