Flow Cytometers and Applications of Flow Cytometry
Flow cytometry is an analytical technique that interrogates cells or other particles suspended in fluid as they pass in single-file through a laser beam. It measures both light scattering and fluorescence emitted by the particles.
The most common application of flow cytometry is immunophenotyping. This involves staining cells with antibodies that bind to specific cell surface antigens.
What is flow cytometry?
Flow cytometry is a powerful cell biology technique that uses laser-based technology to detect and characterize individual cells in a sample. When a cell or particle passes through the interrogation point of a flow cytometer, the interaction with the laser light is measured as forward and side scatter along with fluorescence intensity. A fluorescent label, often a monoclonal antibody conjugated to a fluorophore, is bound to the cell and its detection allows the characterization of specific structures within the cell. This information can be used for multiple biological applications such as immunophenotyping, identifying and quantifying rare or abnormal cells, determining the phase of the cell cycle, and assessing the occurrence of apoptosis.
Immunophenotyping is perhaps the most popular and widely used application of flow cytometry. A well-designed immunophenotyping experiment identifies and quantifies a population of cells using both surface and intracellular antigen staining. Typically, a set of antibodies bind to different cellular antigens in the cell and are defined by their cluster of differentiation (CD) number which allows for unique characterization such as leukemia/lymphoma phenotyping or identification of the T helper cells that support immune responses against infectious agents.
Flow cytometry can also identify apoptosis by detecting the expression of a protein called caspase 3 which is typically elevated in the early stages of apoptosis. Flow cytometry can also detect the presence of autophagy in the cell which is indicative of an active process to break down and recycle cellular components and remove damaged proteins.
Flow cytometry can also determine the number of cells in a population with a density plot which displays not only fluorescence intensities but a relative frequency of events in a specific region of the scatter-plot. This is useful for cell population analysis and sorting.
What is a flow cytometer?
A Flow cytometer is a powerful analytical tool used to identify, characterize, and isolate different populations of cells. The Flow Cytometry and Cell Sorting Facility at Baylor College of Medicine provides the instrumentation, technical expertise, and software needed for multiparameter immunophenotyping and high-speed single-cell sorting in a variety of applications in basic and translational research.
The facility offers both assisted and independent operation of cytometers for a fee based on the type of use requested. All users must be trained by FCCS staff for unassisted operation of cytometers before running any samples on their own. Failure to comply with FCCS policies and procedures may result in the forfeiture of facility privileges.
In addition to its analytic cytometry capabilities, the FCCS has two Becton Dickinson FACS Aria Fusion sorters that operate in a biological safety hood, allowing sorting of live human cells and samples that have been exposed to infectious agents. The FCCS also has three Becton Dickinson LSR II instruments for multi-color flow cytometry analysis, a BioRad Bioplex (Luminex 200s), and a Miltenyi autoMACS.
While flow cytometry allows researchers to isolate cell populations based on the proportion of each type of cell in a pool of heterogeneous cells, cell sorting takes that technology one step further. Sorting uses the results of flow cytometry to direct aerodynamically-powered streams of fluid to separate specific cell types from a larger population.
While fluorescence-based cell sorting has been available for decades — isolating different types of cells by tagging them with antibodies that can glow in different colors, then following an immunostaining protocol to distinguish those cells from others — current sorters have the ability to sort individual cells at a much higher rate and provide more information about the cell population being sorted. In fact, the new BD WOLF (Wide Open Field Array) cell sorter can deliver up to 1,000 times more data than conventional cytometry.
Applications of flow cytometry
Flow cytometry is a versatile analytical tool that can be used for a wide variety of applications. The technique allows researchers to identify and sort cells and particles based on their physical and chemical characteristics. It is also useful for assessing the activity and function of individual cells.
During the analysis process, a sample of cells or particles is suspended in a fluid stream and passed through a laser beam one at a time. The particles and cells are labeled with fluorescent markers that absorb and emit specific bands of wavelengths. The scattered and emitted light is then captured by optical components and analyzed by software to generate data on the sample. This data is usually presented as a series of histograms or dot plots that show the physical and chemical properties of each particle in the sample.
The most common application of flow cytometry is immunophenotyping, which is the process of identifying and quantifying the presence of specific cell types in a sample. This type of analysis is often used in leukemia and lymphoma diagnosis, as it can help doctors determine the type of cancer that a patient has by measuring the amount of protein in their white blood cells. It is also useful for evaluating immune response, such as when a vaccine is being developed.
Flow cytometry can also be used to analyze the structure of cells and particles, as well as soluble analytes in the sample. For example, a fluorescent calcium indicator dye can be used to assess the level of activation and signaling in a cell. This type of measurement is important because it can help researchers understand how cells communicate with each other and the surrounding environment, which may lead to better understanding of the mechanisms behind cellular reactions and responses.
Working principles of a flow cytometer
A flow cytometer uses an intense light source to create a tightly focused beam that interrogates cells or particles within a sample tube. This allows for multiple parameters to be measured on each particle, without overlapping data. The instrument also contains a number of optical filters that can separate and collect different components of the cell or particle, including light scatter and fluorescence.
A fluorescent dye is often used to stain specific proteins on the surface of the particle or cell. These dyes emit light at a particular wavelength when excited by the laser, which can then be detected by the instrument’s detectors. These detectors can be either photomultiplier tubes (PMTs) or avalanche photodiodes. The PMTs are most common, however, because they can measure a much greater range of fluorescent wavelengths.
Forward and side scatter, as well as fluorescence, are all split into their constituent parts by a series of filters and mirrors within the instrument. Depending on the experiment, the filtering system can select a set of parameters to collect. This can include the ability to identify distinct populations based on their size and granularity (as an example, large granulocyte cells have a high FS but low SS), or if they are positive for a specific protein of interest.
Using a process called cell sorting, the sorted cells can be collected in a tube or plate for further testing and analysis. To perform this operation, the cells or particles must first be converted into a single-cell suspension by enzymatic digestion or mechanical dissociation. This ensures that each individual cell is identified and characterized for its own set of parameters. Finally, the resulting data can be presented in either histograms or dot-plots to allow for rich, multiparametric data analysis.
Types of flow cytometers
There are a number of different types of flow cytometers. Each instrument is designed for a particular type of cellular analysis or sorting. Traditional cytometers analyze a sample by passing it through the cytometer one cell or particle at a time. As they move through the instrument, each cell or particle is measured for various characteristics based on its light scattering and fluorescence properties. The resulting data is then used to identify and classify each cell or particle.
To do this, the cytometer uses sheath fluid to hydrodynamically focus each cell or particle through a small nozzle at a high velocity. The nozzle also serves to measure forward and side scatter (SS and FS). In some instruments, fluorescence detectors can also be used. These detectors detect the emitted light from fluorescently stained proteins or chromophores attached to each cell or particle.
Each emitted light signal is read by a set of sensors, usually photomultiplier tubes or avalanche photodiodes. The photodiodes or PMTs convert the emitted light into an electronic signal that is recorded on a computer for analysis. The signal is then analyzed to determine the various characteristics of each cell or particle such as its size, granularity, apoptosis, or intracellular calcium flux.
A very useful application of this technique is immunophenotyping. This allows the identification of specific cells within a mixed population such as T helper or cytotoxic T cells or antigen-specific T cells. This provides a valuable tool for understanding immune response and for monitoring diseases such as autoimmunity, cancer, and leukemia.
Flow cytometry can simultaneously measure the physical and chemical characteristics of thousands of cells or particles per second. This information can be used to identify and distinguish between cells based on their unique phenotype or function and can also be used to select (gate) a subset of the cells for further processing such as culturing, microscopy, or immunohistochemistry.
