What is Flow Cytometry? – A complete guide to Flow Cytometric Analysis

Flow cytometry is a powerful technique for analyzing the physical and chemical properties of cells or particles in a liquid as they pass through single or multiple lasers. It is used in both, research and clinical laboratories, to quickly analyze e.g. size, characteristics, type of cell or state of cell cycle and cell proliferation by using fluorescence. Therefore, a scientist treats the cells with fluorescent antibody to label (or “tag”) specific proteins on the surface or with fluorescent dyes inside of cells.

How does Flow Cytometry work?

In flow cytometry, a liquid cell suspension is passed through a laser system and a sensor detects the light refracted or emitted by the cells. Each cell type has some characteristic features that make it unique, such as the proteins on the cell surface ,the type of organelles in the cells or the metabolism and DNA synthesis. Depending on the scientist’s interest, the researcher uses certain markers such as fluorescent antibodies to mark (or “tag”) certain proteins on the surface, or fluorescent dyes to mark newly synthesized DNA inside the cells. The principle of detection is based on the scattering of light and fluorescence to detect certain characteristics of the cells such as size, complexity and the presence of certain markers.

Light Scattering & Fluorescence Detection

Light scattering: When the cells pass through the laser, the light is scattered in different directions. Forward scattering (FSC) provides information about the size of the cells, while side scattering (SSC) reveals the complexity or granularity of the cells.
Fluorescence detection: Fluorescent dyes are bound to specific cell components like DNA and enable the identification of cell viability and proliferation providing information about cytotoxity of e.g. drugs. Assays can report either total or live cell numbers, or measure DNA synthesis in single cells. We offer dyes and assays to track proliferation.

Cell sorting using Flow Cytometry (FACS)

How FACS works: Fluorescence-activated cell sorting (FACS) is a special form of flow cytometry. It does not only analyzes the cells, but can also sort and separate them based on their physical or fluorescent properties to collect them in different vails for further experiments and analysis.
FACS is often used to sort cells for research or clinical applications, e.g. to isolate specific cells or cell types for further investigation or to separate cancer cells for better diagnosis.
Applications of FACS analysis:

Immunophenotyping of T-cells – FACS is commonly used to identify and quantify specific T cell populations such as CD4+ and CD8+ T-cells. This is crucial for the study of immune responses, especially in immunotherapy, vaccine development and immune health monitoring.

T cell activation – researchers use FACS to measure the activation status of T-cells by detecting activation markers (e.g. CD25, CD69). This is important for analyzing the response of T-cells to antigens, cytokines or treatments in both clinical and research settings.

T cell proliferation assays – FACS is a powerful tool for monitoring T cell proliferation in response to stimuli. By using proliferation dyes, researchers can track cell division and quantify how many T-cells are actively dividing, which is important for understanding immune responses and developing immunotherapies. Our T cell proliferation assay incoperates EdU in de novo synthezied DNA and tags the DNA with a dye for fluorescence detection via Click Chemistry.

Cell cycle analysis – FACS can determine which phase of the cell cycle T-cells or other immune cells are in (e.g. G0/G1, S phase, G2/M). This is important to investigate how treatments or diseases affect cell division and proliferation.

Apoptosis detection – FACS helps to measure apoptosis in T-cells by detecting markers such as annexin V and propidium iodide. This is critical for assessing how therapies induce cell death in abnormal or dysfunctional immune cells.

Isolation of T cell subsets – FACS enables the isolation of specific T cell subsets based on fluorescent markers. This is particularly useful for downstream applications such as gene expression profiling or functional assays that focus on a purified cell population.

Monitoring immune responses – FACS can be used to track the expansion of specific T-cell clones during an immune response, particularly in cancer immunotherapy, helping to assess the efficacy of treatment.

Applications of Flow Cytometry

Flow cytometry is widely applied in many fields, including:

Immunology and cell biology

In the fields of immunology and cell biology, flow cytometry plays an important role in the study of cell proliferation and immune response. Our EdU-based proliferation assays provide a robust method for labeling newly synthesized DNA and tracking cell division. These assays are particularly useful for analyzing T cell proliferation, making them ideal for immune function and drug development studies.

For example, researchers can use our EdU-based assays to monitor the proliferation of T cells after activation, similar to the widely used CellTrace™ kits, but with greater specificity and sensitivity in detecting DNA synthesis. This technique allows for more accurate quantification of proliferating cells and helps to understand how immune cells respond to different stimuli or treatments.

Cancer research

In cancer research, flow cytometry is often used to study tumor cell growth and the response of cancer cells to therapies. Our EdU-based proliferation assays offer a powerful alternative to conventional methods by precisely labeling and tracking the replication of cancer cells. By fluorescently labeling DNA synthesis, researchers can monitor how fast cancer cells are dividing, which is crucial for understanding tumor growth and response to therapy.

Clinical diagnostics

In the realm of clinical diagnostics, flow cytometry is used to monitor disease progression and evaluate the proliferation rates of cells. Our EdU-based assays offer clinicians a more refined tool for tracking DNA synthesis in rapidly dividing cells, providing insights into conditions like hematological malignancies or immune deficiencies. By enabling the detailed analysis of cell proliferation in patient samples, this technology is becoming a vital asset in personalized medicine and tailored treatment strategies.

As demonstrated in recent publications (e.g., doi.org/10.1016/j.jim.2022.113228), our EdU-based proliferation assays are a competitive alternative to CellTrace™ kits for T-cell analysis, making them an invaluable tool in both research and clinical applications.

This approach highlights your key products in relation to the broader fields of immunology, cancer research, and clinical diagnostics, while showcasing their advantages over competing technologies like CellTrace™ kits.

Flow Cytometry data analysis

Flow cytometry is a widely used technique to analyze physical and chemical properties of cells or particles as they pass through a laser. It is commonly used to assess cell populations based on parameters like size, complexity, and fluorescence intensity. In this example, we are analyzing HeLa cells that have been incubated with the nucleoside analog EdU (5-ethynyl-2’-deoxyuridine) for 2 hours, which incorporates into newly synthesized DNA during the S-phase of the cell cycle. A click chemistry reaction is then performed, labeling the EdU with the fluorescent dye 6-FAM, an analog of Alexa Fluor 488, allowing for detection via flow cytometry.

Figure A: Histogram

X-axis (Fluorescence Intensity Alexa Fluor 488 Channel): The x-axis displays the fluorescence intensity of the cells, indicating the level of EdU incorporation. The numbers on this axis are displayed logarithmically, ranging from to , meaning 100 to 100,000 fluorescence units. This logarithmic scale is standard in flow cytometry because it allows for a wide range of intensities to be visualized, making it easier to distinguish between different cell populations.

The first peak, located between 10²and 10³, represents cells that did not incorporate EdU, indicating these cells were not synthesizing new DNA and therefore have not been in the S-phase of the cell cycle (non-proliferating cells). These cells have very low fluorescence because they did not incoperate the 6-FAM label.

The second peak, which is shifted towards the right (between 10⁴ and 10⁵), corresponds to cells that have actively synthesized DNA and thus incorporated EdU. The shift in fluorescence intensity occurs because the newly synthesized DNA is now labeled with 6-FAM, which emits a fluorescent signal detectable by flow cytometry. The greater the fluorescence intensity, the more EdU was incorporated, indicating higher levels of DNA synthesis and thus active cell proliferation.

Y-axis (Cell Count): The y-axis represents the number of cells at each fluorescence intensity level. The values (0, 60, 100, 160, 200, 260) show how many cells have a specific level of fluorescence. The peak heights correspond to the relative number of cells in each population. The first peak (low fluorescence) corresponds to non-proliferating cells, while the second peak (high fluorescence) corresponds to proliferating cells. The gap between these peaks represents the distinction between non-proliferating and proliferating cells, as determined by the amount of 6-FAM incorporated into their DNA.

Figure B: Scatter Plot

X-axis (Forward Scatter, FSC-A): Forward scatter (FSC) correlates with cell size. Larger cells scatter more light in the forward direction, so the higher the value on the x-axis, the larger the cell. In this plot, cells are spread across different sizes, but the majority fall within a consistent size range.
Y-axis (Side Scatter, SSC-A): Side scatter (SSC) correlates with cell complexity or granularity, which can reflect the amount of internal structures or organelles within the cell. Cells with more complex internal structures, such as granules or vesicles, will have higher side scatter values.

In this scatter plot, you can see two distinct clusters of cells. The upper cluster represents proliferating cells, which are generally larger and more complex due to increased metabolic activity and DNA replication. The lower cluster represents non-proliferating cells, which are smaller and less complex. The separation between these two populations is clear, indicating a successful distinction between cells that are undergoing DNA synthesis (and thus incorporated EdU) and those that are not.

Why These Axes and Scales?

Logarithmic Scale on X-axis (Histogram): In flow cytometry, fluorescence intensity often varies over several orders of magnitude. The logarithmic scale allows for a more meaningful representation of both dim and bright populations on the same plot. Without this scale, lower-intensity signals would be compressed, and higher-intensity signals would dominate the graph, making it difficult to distinguish between different populations.
Peaks and Fluorescence Shifts: The first peak, with cells between 10²and 10³, represents cells that have not synthesized DNA (non-proliferating), as they did not incorporate the EdU and thus have minimal fluorescence. The shift toward higher fluorescence (between 10⁴ and 10⁵) in the second peak indicates successful EdU incorporation into the DNA of proliferating cells, which leads to higher fluorescence due to the 6-FAM labeling. This shift shows the difference between cells in active DNA synthesis and those that are not.

Flow Cytometry software for data interpretation

Several software tools are available to analyze flow cytometry data analysis. Common options include:

FlowJo: One of the most widely used software for flow cytometry data analysis, offering comprehensive tools for gating, visualizing, and interpreting cell populations.
FACSDiva: This software is commonly paired with BD Biosciences flow cytometers and provides powerful data acquisition and analysis capabilities.
Kaluza: Offered by Beckman Coulter, Kaluza is known for its user-friendly interface and advanced tools for visualizing complex data sets.
FCS Express: Another powerful option, offering features that allow publication-ready graphics, advanced data analysis, and reporting tools.

These software programs provide functionalities like gating (defining cell populations based on specific markers or parameters), overlaying histograms, and performing statistical analysis to quantify and interpret cell populations.

Interpretation of the Data

The histograms and scatter plots in this experiment allow researchers to identify and quantify the population of proliferating cells:

Proliferating Cells: These cells show high fluorescence in the histogram and are represented in the upper population in the scatter plot. The EdU incorporation, followed by 6-FAM labeling, indicates that these cells are actively synthesizing DNA.
Non-Proliferating Cells: These cells have low fluorescence in the histogram and are located in the lower population in the scatter plot, indicating no significant DNA synthesis.

By using these plots, researchers can measure the percentage of proliferating cells, assess the effectiveness of treatments that influence cell division, and investigate cell cycle dynamics. The ability to distinguish between proliferating and non-proliferating cells is crucial for applications in cancer research, drug development, and immunology.

Importance of Flow Cytometry in research and medicine

Flow cytometry has become a cornerstone in modern research and clinical practice due to its ability to quickly and accurately analyze multiple parameters of individual cells in a single run.

Diagnosing diseases and personalizing treatments

Flow cytometry allows monitoring of disease progression by tracking changes in cell populations over time. For example, in cancer, it helps assess tumor heterogeneity, monitor minimal residual disease, and evaluate treatment response. In HIV/AIDS, it tracks CD4+ T-cell counts to guide antiretroviral therapy. In autoimmune diseases, it identifies aberrant immune cell subsets.

More Applications:

Immunophenotyping: Flow cytometry is widely used for immunophenotyping of various specimens, including whole blood, bone marrow, serous cavity fluids, cerebrospinal fluid, urine, and solid tissues.
Cell Size and Complexity: It can measure cell size, cytoplasmic complexity, DNA/RNA content, and various membrane-bound and intracellular proteins.
Cytokine Detection: Flow cytometry aids in diagnosing diseases by analyzing cytokine detection.
Protein Analysis: It helps identify abnormal protein expression patterns associated with diseases.
Cell Cycle and DNA Analysis: Flow cytometry provides insights into cell cycle progression and DNA content.
Viral and Bacterial Cell Counts: It can quantify viral and bacterial populations.

Flow Cytometry Protocol

Typically, flow cytometry experiments follow these basic steps:

1.) Sample Preparation:

Create a single-cell suspension from your sample. This can be done by disaggregating solid tissues (mechanically or enzymatically) or using non-adherent cells from culture. If you need to study intracellular components, permeabilize the cell membrane to allow dyes or antibodies to penetrate while maintaining overall cell integrity.

Staining and Antibodies:

Stain the cells with fluorophore-labeled antibodies specific to cellular markers of interest. For cell surface markers, perform sequential staining if needed (stain for surface markers first, then fix, permeabilize, and stain for intracellular targets). Optimize antibody concentrations and choose appropriate controls (e.g., isotype controls, fluorescence minus one controls) to ensure accurate results.

Flow Cytometer Setup:

Load the stained sample into the flow cytometer. The fluidics system directs cells in single file past an interrogation point. Lasers focus on the cells, scattering laser light, and bound fluorophores emit fluorescent signals. Photomultiplier tubes (PMTs) detect and amplify the emitted light, converting it into measurable electrical signals.

Data Acquisition and Analysis:

Collect data on forward scatter (FSC) and side scatter (SSC) characteristics to identify distinct cellular populations. Use appropriate experimental controls to analyze the data. Gate cells of interest based on size, shape, complexity, and marker expression.

Quality Control and Settings:

Confirm flow cytometer settings (voltages, compensation, etc.). Calculate compensation for spectral overlap. Use post-acquisition analysis software if necessary. Remember that proper sample preparation, accurate staining, and thoughtful controls are critical for successful flow cytometry experiments.

2.) Cell Preparation:

- Start by culturing your cells appropriately (adherent or suspension cells) and ensure they are in the desired growth phase.
- Harvest the cells and wash them to remove any media or serum components.
- Fix the cells using a suitable fixative (e.g., 4% paraformaldehyde in PBS) to preserve their structure.

3.) EdU Labeling:

- Prepare an EdU stock solution (usually 20 mM in DMSO or water).
- Add the EdU stock solution to your cell culture medium at the desired concentration (typically 10-50 μM) and incubate the cells for a specific time (usually 1-4 hours).
- EdU (5-ethynyl-2’-deoxyuridine) is incorporated into DNA during active DNA synthesis. It serves as a nucleoside analog to thymidine.
- Unlike BrdU assays, which require DNA denaturation, EdU labeling is compatible with mild fixation and permeabilization methods.

4.) Fixation and Permeabilization:

- Fix the cells using a fixative (e.g., paraformaldehyde).
- Permeabilize the cells using a permeabilization buffer (e.g., 0.1% saponin, 0.002% NaN3, 1% BSA in PBS).

5.) Click Reaction:

- Prepare the click reaction components:
  - Copper catalyst (CuSO₄, THPTA).
  - Reducing agent (sodium ascorbate).
  - 6-FAM or 6-FAM-Picolyl-Azide (a fluorescent azide).
- Add the click reaction components to the permeabilized cells.
- The click reaction between the azide in EdU and the alkyne in the AFDye Picolyl Azide results in covalent binding of the fluorescent dye to the incorporated EdU.

6.) Staining and Flow Cytometer Setup:

- Wash the cells to remove excess reagents.
- Stain the cells with additional markers (if desired) for multiplexing.
- Analyze the stained cells using flow cytometry.
- Load the stained sample into the flow cytometer.
- The fluidics system directs cells in single file past an interrogation point.
- Lasers focus on the cells, scattering laser light, and bound fluorophores emit fluorescent signals.
- Photomultiplier tubes (PMTs) detect and amplify the emitted light, converting it into measurable electrical signals
- Detect the fluorescence signal from the incorporated EdU using appropriate filters (e.g., Pacific Blue™, Alexa Fluor® 488, or Alexa Fluor® 647)

7.) Data Acquisition and Analysis:

- Collect data on forward scatter (FSC) and side scatter (SSC) characteristics to identify distinct cellular populations
- Gate cells of interest based on size, shape, complexity, and marker expression.
- Determine the percentage of cells in the S-phase (actively synthesizing DNA) based on the EdU signal.
- Compare the results with untreated control cells or cells treated with other conditions.

8.) Quality Control and Settings:

Remember that EdU-based assays offer advantages over BrdU assays, including compatibility with cell cycle dyes and the ability to multiplex with other antibodies. These assays are valuable for assessing cell proliferation, genotoxicity, and drug effects.

Flow Cytometry reagents and equipment

Reagents and Kits:

ClickTech EdU Flow Cytometry Assay Kits: These kits use 5-ethynyl-2’-deoxyuridine (EdU) as a nucleoside analog to thymidine. EdU is incorporated into DNA during active DNA synthesis. Detection is based on a click reaction, allowing efficient detection without the need for DNA denaturation.

BrdU Assays: bromodeoxyuridine (BrdU) assays involve the incoperation of BrdU as thymidine analog into DNA. BrdU is detected by specific antibody-which requires the denaturation of DNA.

Cell Proliferation dilution Kits: These kits allow you to permanently label cells with fluorescent stains for tracking generations of cell division.

Markers:

Ki-67 Antibody: Detects a nuclear protein expressed in proliferating cells.

MCM2 Antibody: Identifies another nuclear protein expressed during cell proliferation.

PCNA Antibody: Detects proliferating cell nuclear antigen.

Multiplexing: EdU labeling is compatible with cell cycle dyes and can be multiplexed with antibodies against other markers.

Flow Cytometry Equipment:

Flow Cytometer: The core instrument for flow cytometry. It analyzes cells in a fluid stream, measuring their physical and chemical properties.

Lasers: Provide excitation light for fluorochromes. Common lasers include blue (488 nm), red (633 nm) or orange (594 nm).

Optics: Collect and filter emitted fluorescence signals.

Detectors: Capture fluorescence emissions at specific wavelengths.

Software: Analyze data and create plots.

Sample Preparation Equipment:

Centrifuge: For cell separation and sample preparation.

Cell Strainer: Removes debris and aggregates.

Pipettes and Tubes: For handling samples.

Refrigerator and Freezer: Store reagents.

Safety Equipment: Lab coat, gloves, and eyewear.

Cell Culture Equipment (if working with live cells):

CO2 Incubator: Maintains cell culture conditions.

Laminar Flow Hood: Provides sterile environment for cell handling.

Microscope: Inspect cell cultures.

Cell Culture Plates/Dishes: For growing cells.

Choosing the right Flow Cytometer for your needs

Types of Flow Cytometers:

- Analytical Flow Cytometers: Designed for high-throughput analysis of cells and particles, they measure various parameters such as cell size, granularity, and fluorescence intensity. Ideal for applications like immunophenotyping, cell viability assays, and intracellular protein analysis.
- Cell Sorters (FACS): Not only analyze but also physically separate cells based on their properties. Crucial for applications requiring pure populations of specific cell types, such as stem cell research, cancer research, and single-cell genomics.
- Benchtop Flow Cytometers: Compact and suitable for laboratories with limited space. Despite their smaller size, they offer robust analytical capabilities and are suitable for clinical diagnostics, environmental testing, and routine laboratory assays.
- Portable Flow Cytometers: Lightweight and designed for field or point-of-care testing. Ideal for applications such as infectious disease diagnosis, water quality testing, and agricultural monitoring.
- High-Throughput Flow Cytometers: Equipped with advanced automation features to handle large sample volumes efficiently. Essential for large-scale studies, drug screening, and clinical trials.

Key Features to Consider:

- Number of Parameters: More parameters allow for complex multiparametric analysis, essential for detailed cellular profiling and biomarker studies.
- Sensitivity and Resolution: Critical for detecting low-abundance markers and distinguishing between closely related cell populations.
- Ease of Use: Look for user-friendly systems with intuitive software interfaces and automated features.
- Throughput: Consider the number of samples processed in your lab and choose a flow cytometer with appropriate throughput capabilities.
- Compatibility with Assays: Ensure that the flow cytometer meets the sensitivity and specificity requirements of your lab’s assays.

Fluorophore Selection:

- When designing panels, consider the available fluorophores and their spectral properties. Spectral flow cytometry allows discrimination of unique spectral signatures, enabling compatibility and distinction of many fluorescent combinations.
- Understand the laser configurations and optical sensitivity of your instrument to choose appropriate fluorochrome combinations

Flow Cytometry – Guide to analysis, applications & protocols