Flow cytometry

Flow Cytometry

A flow cytometry tube used for sample preparation.

Flow cytometry is a powerful analytical technique used to measure the physical and chemical characteristics of cells or particles as they flow in a fluid stream through a beam of light. This method is widely used in various fields such as immunology, pathology, and molecular biology for cell counting, cell sorting, biomarker detection, and protein engineering.

Principle of Flow Cytometry

Flow cytometry works by suspending cells in a stream of fluid and passing them through an electronic detection apparatus. As each cell passes through the laser beam, it scatters light and may emit fluorescence if it has been labeled with a fluorescent marker. The scattered light is detected by photodetectors, which convert the light signals into electronic signals that can be analyzed by a computer.

Diagram of a flow cytometer.

The key components of a flow cytometer include:

• Fluidics system: Directs the flow of cells in a single file through the laser beam.
• Optics system: Consists of lasers and lenses to focus the light on the cells and collect the emitted light.
• Electronics system: Converts the light signals into electronic signals for analysis.

Applications of Flow Cytometry

Flow cytometry is used in a variety of applications, including:

• Immunophenotyping: Identifying and quantifying different types of cells in a heterogeneous population, such as different types of white blood cells in blood samples.
• Cell sorting: Separating cells based on their properties, such as size, granularity, and fluorescence intensity.
• Apoptosis detection: Measuring the process of programmed cell death by detecting changes in cell membrane integrity and DNA fragmentation.
• Cell cycle analysis: Determining the distribution of cells in different phases of the cell cycle.

Data Analysis

Data from flow cytometry is typically displayed in histograms or dot plots. A histogram shows the distribution of a single parameter, while a dot plot can show the relationship between two parameters. Gating is a process used to select specific cell populations for further analysis.

Example of flow cytometric gating using side scatter and CD45.

Advantages and Limitations

Flow cytometry offers several advantages, including rapid analysis of thousands of cells per second, the ability to analyze multiple parameters simultaneously, and high sensitivity and specificity. However, it also has limitations, such as the need for specialized equipment and expertise, and the potential for artifacts if samples are not prepared correctly.

Also see

• Cell sorting
• Immunophenotyping
• Fluorescence-activated cell sorting
• Laser
• Biomarker

References

• Shapiro, H. M. (2003). Practical Flow Cytometry. Wiley-Liss.
• Givan, A. L. (2001). Flow Cytometry: First Principles. Wiley-Liss.