An Introduction to Flow Cytometry

What is flow cytometry?

This technique allows researchers to rapidly identify and analyze specific cell types, monitor changes in cell populations over time and study the behavior of cells under various conditions. It is widely used in fields such as immunology, hematology, oncology and microbiology and has become an essential tool for researchers and clinicians alike. Flow cytometry can accurately measure multiple parameters of thousands of cells or particles in just a few seconds using lasers and detectors.

How does flow cytometry work?

Flow cytometry works by suspending cells or particles in a liquid and passing them through a narrow, focused stream of fluid, typically sheathed by another fluid, such as saline or phosphate-buffered saline (PBS). As the cells or particles flow through the stream, they pass through a series of lasers and detectors that measure various properties of the sample.

The first step in flow cytometry is sample preparation in which cells or particles are collected from a biological sample and purified.

The purified sample is then stained with fluorescent dyes, which can target specific molecules or structures within the cell. These dyes can provide information about cellular features, such as cell surface markers, intracellular proteins and DNA content.

After the sample is prepared, it is introduced into the flow cytometer. The sample is drawn into the instrument by a syringe or pump and then focused into a narrow stream using a flow cell or nozzle. The stream of cells or particles passes through one or more lasers, which excite the fluorescent dyes and cause them to emit light at specific wavelengths.

The emitted light is collected by detectors, which measure the intensity and wavelength of the fluorescence. Depending on the instrument, multiple lasers and detectors may measure multiple fluorescent signals simultaneously. In addition, other detectors can measure physical properties of the cells, such as size and granularity.

As the cells pass through the flow cytometer, they are subjected to a series of electrical charges that cause them to separate from each other, based on their physical and chemical properties. This separation process, known as electrostatic deflection, can be used to sort the cells or particles into different populations based on the parameters measured by the lasers and detectors.

Finally, the data generated by the flow cytometer is analyzed using specialized software, which can display the data in various formats, such as histograms, dot plots or contour plots. These plots can identify and analyze specific cell populations, measure the expression of specific markers and track changes in the population over time or in response to different treatments.

Overall, flow cytometry provides a powerful and flexible tool for studying the properties of individual cells or particles in complex biological samples. By combining fluorescent labeling with advanced optics and electronics, flow cytometry allows researchers to rapidly and accurately analyze complex cellular processes and gain insight into the function and behavior of individual cells.

Introduction to the flow cytometer instrument

A flow cytometer instrument uses lasers, optics and electronics to measure and analyze cells or particles in a sample. It has the following components:

Fluidics System

The fluidics system of a flow cytometer is responsible for preparing and transporting the sample through the instrument. It includes a sample injection port, a sheath fluid system and a flow cell or nozzle that focuses the sample into a narrow stream.

The fluidics system also regulates the pressure, flow rate and velocity of the sample, to ensure that the cells or particles pass through the laser beam in a controlled and consistent manner.

Laser System

The laser system of a flow cytometer typically includes one or more lasers that emit light at specific wavelengths. The lasers are selected based on the fluorescent dyes used to label the sample and are typically in the visible or near-infrared range.

The lasers are aligned to intersect with the stream of cells or particles, causing the fluorescent dyes to emit light at specific wavelengths, which is then detected by the optics system.

Optics System

The optics system of a flow cytometer includes a series of filters and lenses that direct and collect the emitted light from the fluorescent dyes. The light is separated into different wavelengths using filters and then focused onto photomultiplier tubes (PMTs) or other detectors which convert the light into an electrical signal.

The detectors measure the intensity of the fluorescent signal at specific wavelengths, providing information about the quantity and quality of the labeled molecules or structures in the cell.

Electronics System

The electronic system of a flow cytometer includes the hardware and software that controls the instrument, collects the data from the detectors and processes the data into usable information. The electronics system may include digital signal processing circuits, data acquisition cards and specialized software that allow users to view and analyze the data in various formats.

Sorting System

Some flow cytometers have a sorting system, which uses electrostatic deflection to sort cells or particles into different populations based on their physical and chemical properties.

The sorting system typically includes one or more collection chambers, a high-voltage power supply and an electrode system that generates an electrical field to deflect the cells or particles into the appropriate collection chamber.

Flow cytometers are a sophisticated and versatile tool for studying the properties of individual cells or particles in complex biological samples. Its many components work together seamlessly to generate high-quality data and provide insights into the behavior and function of cells in various biological contexts.

What are the signals in flow cytometry?

Flow cytometry generates three types of output signals that provide information about the physical and chemical properties of cells or particles in a sample. These output measurements include forward scatter (FSC), side scatter (SSC) and fluorescence signals. That said, these three signals are described in detail below.

Forward scatter (FSC)

Forward scatter (FSC) is one of the three output signals flow cytometry generates. It measures the size of a cell or particle based on the amount of light scattered in the forward direction as it passes through the laser beam. Larger cells or particles scatter more light in the forward direction, producing higher FSC signals. FSC provides important information about the physical characteristics of cells or particles, such as their size, shape, and complexity. It commonly identifies and characterizes different types of cells or particles in a sample based on their size and shape.

For example, red blood cells are typically smaller and more uniform than white blood cells , which are larger and more varied in shape. By measuring the FSC signals of cells in a sample, researchers can distinguish between different cell populations and quantify their relative quantity.

In addition to providing information about cell size, FSC can also tell about the complexity and granularity of cells. For example, cells with more internal complexity, such as those with many organelles or granules, may scatter more light in the forward direction and produce higher FSC signals. That can be used to identify cells with different structures, such as lymphocytes, monocytes and neutrophils.

Side scatter (SSC)

Side scatter (SSC) is another output signal generated by flow cytometry. It measures the complexity or granularity of a cell or particle based on the amount of light scattered at an angle to the laser beam. Cells that have a complex structure with more granules scatter more light at an angle and produce higher SSC signals than cells that have a less complex structure.

SSC can reveal the internal characteristics of cells or particles, such as their composition, structure and function. It identifies and characterizes different types of cells or particles in a sample based on their internal structure and complexity.

For example, immune cells such as lymphocytes and neutrophils have different internal structures and functions, which can be distinguished based on SSC signals. Different subpopulations of cells, such as activated or resting immune cells, can also be identified based on their SSC signals. In addition,

SSC can provide insights into the composition and function of cells. SSC can be used to measure a cell's granularity or lysosomal content, indicating its activation status or function. Similarly, SSC can distinguish between live and dead cells in a sample, based on the cell's internal complexity.

Fluorescent signal

A fluorescent signal is an output signal generated by flow cytometry. It measures the fluorescent properties of cells or particles labeled with fluorescent dyes or antibodies. When the laser excites, the dyes emit light at specific wavelengths, which the optics system detects and converts into a fluorescent signal. That fluorescent signal provides information about the molecular properties and functional characteristics of cells or particles in a sample.

Fluorescent signals can identify and characterize different types of cells or particles in a sample based on their molecular properties and functional characteristics.

For example, fluorescently labeled antibodies can identify specific cell surface markers or intracellular molecules, such as cytokines, transcription factors, or cell cycle proteins. By measuring the fluorescent signal of cells in a sample, researchers can distinguish between different cell populations and quantify them.

The fluorescent signal can also be used to measure the functional characteristics of cells. For example, fluorescent dyes measure calcium flux, or reactive oxygen species production, which indicates cellular metabolism and signaling pathways.

What is flow cytometry used for?

You might be wondering What is flow cytometry used for? Flow cytometry can be used in a variety of research and clinical applications. Here are some of the most common applications of flow cytometry:

Immunophenotyping

In immunophenotyping, flow cytometry can identify and characterize different cell populations based on their surface markers.The technique can also be used to diagnose diseases, monitor immune system function and track disease progression.

Cell Cycle Analysis

Flow cytometry can be used to measure the DNA content of cells to determine their cell cycle phase. It can also be used to study cell proliferation and identify abnormalities in cell cycle regulation.

Apoptosis Detection

Flow cytometry can be used to measure changes in cell size, shape and DNA content to identify apoptotic cells, and to help It is used to determine the efficacy of anti-cancer drugs.

Intracellular Protein Analysis

Flow cytometry measures intracellular protein levels to study signaling pathways and post-translational modifications. In addition, it can be used to identify drug targets and evaluate the efficacy of drug treatments.

Functional Assays

Flow cytometry can measure cellular function, such as cytokine secretion, phagocytosis and oxidative burst, to study immune system function and disease pathogenesis.

Microbial Analysis

In microbial analysis, flow cytometry can identify and quantify bacterial, viral and fungal cells in a sample.It can also b used to diagnose infectious diseases and monitor microbial communities in environmental samples.

Cell Sorting

Flow cytometry can be used to separate cells based on their physical and chemical characteristics, or to isolate and purify rare cell populations to generate cell lines.

Learn more about flow cytometry

Flow cytometry can be a powerful and flexible tool for studying cells and particles in biological and clinical samples. Learn more about flow cytometry solutions from Avantor.