What Is Flow Cytometry & How Do Flow Cytometers Work?

Earth is teeming with biological life. Thousands of animal species, billions of unique human beings, and lands with diverse geographies fill the place we call home. Much of this diversity stems from the assortment of cells that work together to sustain life.

Cell populations can contain individuals with spontaneous mutations, at different cell cycle phases, and with random gene expression. Profiling these living cells can reveal much about how our bodies operate. Recognizing this, researchers developed tools to advance molecular and cell biology research.

These efforts began with microscopy. But living cell populations within a sample can number in the billions. Hence, researchers developed high-throughput technologies that could comprehensively characterize these cells. That’s where flow cytometers and cell sorters come in.

These innovative machines observe individual cells and sort them by their traits. After sorting these populations, researchers can isolate and experiment with them further. Both tools are indispensable for the differentiation of single cells in a short time frame. Nonetheless, operating these machines and assuring quality control remains an intimidating task. Knowing how these technologies work and what flow cytometers and cell sorters to select will help you operate these machines safely and effectively.

This article will cover various aspects of flow cytometry and cell sorting, and show how Excedr can help you acquire the equipment you need for characterizing cells with single-cell resolution.

How Do Flow Cytometers & Cell Sorters Work?

Flow cytometers and cell sorters are optical systems that detect subsets of cells with specific characteristics. They facilitate cell characterization by identifying a cell’s or particle’s physical and biochemical traits. Below are some of the characteristics that these machines can measure:

• Size and shape: Flow cytometers determine a cell’s morphology by determining light scattering as they scan a cell. Their lasers can conduct two types of flow cytometric scanning: forward scatter (FSC) and side scatter (SSC). FSC measures how much the laser beam scatters along its path, while SSC measures scatter perpendicular to the laser beam. FSC increases as the cell’s size increases, while SSC increases with increasing granularity, which occurs when cells have more organelles and other intracellular components.
• Intracellular molecules: Flow cytometers can also measure the concentrations of different biomolecules inside cells. For instance, flow cytometers can facilitate the cell analysis of DNA content in plants. This data will help researchers identify ploidy, cell cycle stage, and other DNA-based traits.
• Extracellular molecules: Flow cytometers can also measure the molecules expressed at cell membranes, such as receptors. They can profile transmembrane proteins (proteins located on cell membranes) that bind to different molecules such as cytokines. These features can help researchers distinguish between apoptosis and necrosis based on membrane protein expression.

Flow cytometers and cell sorters are amazing for their ability to profile multiple parameters at the same time. So how do flow cytometers and cell sorters facilitate multiparameter analysis? All flow cytometers and cell sorters contain three components that can quantify multiple traits at once. They are the:

• Fluidics system: The fluidics system takes the cells from cell suspensions and moves them to be detected by a laser. To do this, flow cytometers first inject the cell suspensions into a central core and focus them into a single file with the sheath fluid, a buffered saline solution. This movement is accomplished with the Bernoulli Effect to perform hydrodynamic focusing. Through this phenomenon, flow cytometry data of individual cells can be obtained.
• Optics system: The optics system comprises lasers, dichroic filters, photomultiplier tubes (PMTs), or photodiodes. The lasers generate fluorescence-activated cells that emit colored light at specific wavelengths that the flow cytometer detects. The dichroic mirrors direct the emitting light to specific detectors. Then, the bandpass filters determine which wavelengths will be measured. Lastly, PMTs and photodiodes amplify the fluorescent signals for the system to detect more easily. Each component can be set to concurrently measure light intensities at multiple wavelengths.
• Electronics: A flow cytometer and cell sorter’s electronics convert the fluorescent signals into digital signals that their computers can read and analyze. PMTs mediate this conversion by generating a voltage pulse as a cell passes through the laser path. Then, the flow cytometer software creates plots to characterize the intensity of the voltage pulses. These can be a histogram to analyze a single variable, or two variables simultaneously, with a dot plot for cell populations.

Cell sorters have the added function of sorting cells after cell analysis. They typically use electrostatic cell sorting, where the cell sorter uses deflection plates to apply charges to the droplets containing the cells. With user-defined parameters, the cells with specific charges will be sorted into distinct collection tubes that the user can test with downstream assays.

Flow cytometers and cell sorters play a key role in characterizing thousands of individual cells quickly and accurately. Both are important for profiling the diverse cell populations living in complex environments.

Dyes for Integrating Fluorescence into Flow Cytometry

A famous saying goes, “Never judge a book by its cover.” Like books, a cell’s outer appearance does not say everything about its phenotype. Rather, cells possess biomolecules whose activity mediates how a cell behaves in the environment. For researchers, profiling and quantifying these biomolecules is like opening a book to read into the intricate machinery cells possess. The challenge, however, lies in accurately measuring the biomolecules expressed inside the cells and on the cell membranes.

To address this challenge, researchers use three kinds of fluorescent reagents in flow cytometry and fluorescence-activated cell sorting (FACS):

• Fluorescently conjugated antibodies: Fluorescently conjugated antibodies are a staple in immunolabeling. They are the same kinds of antibodies that bind to proteins for confocal fluorescence microscopy and immunohistochemistry. Like those antibodies, fluorescently conjugated antibodies are labeled with fluorophores through covalent binding with thiol groups (S-H) or amine (NH2) groups. After binding to their antigens, fluorescence excitation allows researchers to identify populations based on fluorescence intensity from the conjugated dyes. Fluorescently conjugated antibodies are most commonly used in immunology studies in immunophenotyping assays. In immunophenotyping, proteins expressed on immune cell surfaces, such as T cells, are identified and quantified. These assays can also detect antigens that correspond to different proteins in or on the cell surface.
• Fluorescent dyes: Fluorescent dyes are chemical compounds that light up after being excited by a light source. As stated before, they can be linked with antibodies, but they can also stain cells directly. Fluorescent dyes are most commonly used to assess cell viability. They enter cells and bind to biomolecules to determine whether a cell’s membrane is intact. For instance, DAPI and propidium iodide (PI) are dyes that intercalate with DNA and can identify cells undergoing apoptosis (programmed cell death) and necroptosis.
• Fluorescent proteins: Fluorescent proteins are much larger than fluorescent dyes, being ~25 kD in size. These proteins hold a chromophore at the center of the protein that changes its conformation to enable fluorescence after excitation. Most commonly, proteins of interest are immobilized onto beads and bound with proteins tagged with a fluorescent protein. In this way, protein interactions can be examined at high resolution through flow cytometry.

Each approach leverages the photoelectric effect, where the fluorescent dyes emit light after being excited by a light source at the excitation wavelength. The light from the dyes and proteins is emitted at specific emission wavelengths that flow cytometers and cell sorters can detect.

Flow Cytometry & Cell Sorting Considerations

Irrespective of the reagents used, researchers must keep in mind various factors that affect data analysis. If left unaccounted, the data generated will be compromised. These factors are:

• Autofluorescence: Many biomolecules and cells have intrinsic levels of expression, known as autofluorescence (Read this article to learn more). Additionally, some sets of fluorophores may have overlapping emission spectra, so a signal thought to derive from one fluorophore may come from the other overlapping fluorophore. This phenomenon is known as spectral overlap. Autofluorescence can obfuscate clinical and scientific findings by contributing to false positives when identifying stained immune cell populations. Therefore, researchers need to correct for autofluorescence with methods such as compensation.
• Fluorescent noise: Light emission at low levels is an inherently random process derived from fluorescence and scattered light from flow cells, filters, and lenses in the optical path. For any new flow cytometer, scientists must characterize their instrument closely. One way researchers do this is by measuring a flow cytometer’s signal-to-noise ratio (how much signal is identified relative to technical noise) and its dynamic range (DNR), the minimal and maximum signal strength that the flow cytometers can reliably detect.
• Gating strategy: Gating is one of the most fundamental steps when generating flow cytometry data. Gating is used to sequentially identify and refine cellular populations of interest using fluorescence-based markers. Gating strategies are essential for correctly identifying cells before their fluorescence characteristics are determined. Gating helps researchers differentiate bacterial cells and particulate debris and tells apart immune cells when studying cancer.

Flow Cytometry Applications in Biotechnology & the Clinic

There are many uses of flow cytometry and FACS that researchers have in the clinic. In each instance, researchers need to distinguish between diverse populations of cells that may drive disease in vivo. Researchers have conducted flow cytometer analysis to diagnose and observe the following:

• Cancer diagnosis: Measuring changes in cell populations within a patient can help clinicians diagnose cancer. In fact, clinicians use flow cytometry to diagnose leukemia. The approach relies on detecting the proliferation of abnormal lymphoid and hematopoietic stem cells (cells that mature into red blood cells and immune cells) in the bone marrow. These abnormal cells are others. Such classifications were possible without fluorescence by measuring the FSC and SSC of the lymphocytes.
• Rare disease diagnosis: Researchers have also used flow cytometry to diagnose immune deficiency syndromes. By using fluorescent dyes that bind to immune cell receptors, researchers can identify aberrant increases or decreases in immune cell subpopulations. For example, flow cytometers can detect depleted peripheral B cell counts in X-linked agammaglobulinemia (XLA). The lowered counts prevent people from producing antibodies against infectious disease agents and other antigens.
• Microbiology research: Researchers have also developed flow cytometry and cell sorting for studying microorganisms such as bacteria. Tagging the genes with genes encoding fluorescent proteins such as green fluorescent protein (GFP) can help researchers observe gene expression at single-cell resolution. Flow cytometry analysis has also enhanced the identification of antibiotic-resistant bacteria. The assay can identify bacteria that remain viable after intense antimicrobial treatment. When combined with PCR, scientists can resolve antimicrobial resistance genes and refine treatment approaches.

Flow Cytometer Manufacturers

Excedr can source almost any brand or model of flow cytometer you may be interested in. This is partly because we do not carry an inventory, and partly because we are brand agnostic. Instead of being tied to a specific inventory or list of vendors, you can select the exact manufacturer and model you want to use and we will procure it for you. Some of the instruments we have leased in the past come from brands such as::

• Sysmex: Sysmex is a Japanese company that supplies laboratory testing reagents and testing equipment worldwide. Researchers seeking to sort microscopic cells may find the Cube 6 V2m useful. The Cube 6 V2m is a benchtop flow cytometer designed to analyze microorganisms and microscopic particles. The company also produces flow cytometers for specific uses. The CyFlowTM Ploidy Analyzer helps researchers analyze ploidy in plant cells.
• Beckman Coulter: The company hosts the CytoFLEX Flow Cytometer, which provides 13 bandpass filters to facilitate high-throughput flow cytometry with many kinds of fluorescent dyes. The CytoFLEX can also come with a plate reader modulate to conduct up to 96 assays in a single run. It can also be expanded with the S or LX series and provide more lasers and filter sets for researchers to use.
• Cytonome: The company provides a series of cell sorters that don’t only analyze the cell populations, but separate them into different fractions for further analysis. Making best use of laboratory space is possible with the Cytonome VivaTM, a benchtop cell sorter that can fit within a biohazard hood. For more high-throughput efforts, researchers can also use the Cytonome HydrisTM or the Cytonome GigaSortTM.  

Lease a Flow Cytometer or Cell Sorter with Excedr

Characterizing cells at a single-cell level requires tools that can profile the cells from a sample one at a time. Companies have produced several lines of flow cytometers and cell sorters that can profile a cell’s characteristics and sort them in turn. Such technologies have spurred biomedical research onward, particularly when diagnosing diseases. Nonetheless, purchasing and operating a flow cytometer can be an expensive process.

Excedr’s leasing program can help you establish your lab’s single-cell profiling efforts. We can procure the equipment your lab needs to sort cells and characterize them at a high-throughput rate. From reducing upfront costs to extending your cash runway, speak with our team today to learn exactly how we can help.