What is flow cytometry?

Flow cytometry is a widely used, laser-based method for analyzing the expression of cell surface and intracellular molecules. There are many applications of flow cytometry in research and diagnostics, including simultaneous, multiparameter analysis of single cells and characterizing and defining different cell types in heterogeneous cell populations, such as cancer cells. Flow cytometry assays can also assess the purity of isolated subpopulations, analyze cell size and volume, and sort different cell populations, known as fluorescence-activated cell sorting (FACS)¹. FACS uses a cell sorter, a specialized instrument that sorts cells based on their fluorescent and physical properties, allowing for the collection of sorted cells for further analysis.

A flow cytometry test measures fluorescence intensity produced by fluorescently labeled antibodies specific to proteins on or in cells or ligands that bind to specific cell-associated molecules, such as propidium iodide binding to DNA. The optical system of the flow cytometer is essential for excitation and detection of fluorescence signals.

The staining procedure involves making a single-cell suspension from cell culture or tissue samples. Then, the cells are incubated in tubes or microtiter plates with unlabeled or fluorophore-labeled antibodies and analyzed on the flow cytometer. Antibody staining is a key method for detecting specific antigens in flow cytometry assays.

Multicolor flow cytometry analyzes multiple parameters on thousands of single cells or other particles in seconds^2,3. In multicolor flow cytometry, fluorescent markers like fluorescent proteins are used to characterize and define different cell types of interest in heterogeneous cell populations, assess the purity of isolated subpopulations, and analyze cell size and shape. Surface markers are used to distinguish between different immune cell populations. Fluorescence measurement is central to flow cytometry work, enabling the detection of multiple fluorescence parameters for comprehensive cell characterization.

In this guide, we’ll take you through flow cytometry basics, principles, protocols, and analysis to give you an advanced understanding of how flow cytometry works, when it is valuable, and how to do it. You will also find everything you need to quickly and easily set up your multicolor flow cytometry experiment, from instrumentation basics to recommended controls and data analysis.

If you're looking to get to grips with flow cytometry analysis, check out our free online flow cytometry training.

Flow cytometry instrumentation

Flow cytometry instrumentation forms the backbone of cellular analysis, enabling researchers to rapidly assess and sort cells based on a wide array of physical and chemical characteristics. At the heart of every flow cytometer is the flow cell, a specialized chamber where a single cell suspension is hydrodynamically focused by sheath fluid, ensuring that individual cells pass one at a time through a precisely aligned laser beam. As each cell intersects the laser, fluorescent markers, like fluorescently labeled antibodies, fluorescent dyes, or even green fluorescent protein, are excited, and the resulting fluorescence and light scatter are detected by sensitive photomultiplier tubes or photodiodes.

Modern flow cytometers are equipped with multiple lasers and advanced optical systems, allowing for simultaneous detection of numerous fluorescence parameters. This capability is essential for multicolor flow cytometry panels, which can distinguish between diverse cell phenotypes within heterogeneous cell populations. Imaging flow cytometry takes this a step further by combining the quantitative power of traditional flow cytometry with high-resolution imaging, enabling detailed cell cycle analysis, assessment of cell proliferation, and even visualization of subcellular structures within the entire cell.

Sample preparation is a critical step, typically involving the creation of a single cell suspension from blood samples, bone marrow, or tissue. Fluorescent markers are used to label specific cell surface proteins or intracellular targets, allowing for the identification and characterization of immune cells, tumor cells, central effector memory cells, exhausted T cells, and other important cell subsets. The flow cytometer’s data analysis software then interprets the complex flow cytometry data, using gating strategies to define cell populations, assess cell health and viability, and measure protein expression or gene expression levels.

One of the most powerful features of modern flow cytometers is their cell sorting capabilities. Through fluorescence activated cell sorting (FACS), the instrument can physically sort cells based on their fluorescence characteristics. As single cells pass through the flow cell, those matching specific criteria are encapsulated in charged droplets and deflected into separate collection tubes. This enables researchers to purify cells for further analysis, such as molecular biology studies, cell culture, or functional assays, and is invaluable in both basic research and clinical applications.

How does a flow cytometer work?

When running a cell suspension through a flow cytometer, sheath fluid hydrodynamically focuses cells to get them to pass in a single file through a small nozzle. The resulting tiny stream of fluid takes cells one at a time past a laser light, as shown in Figure 1.¹

Figure 1. Diagram showing an overview of the flow cytometer. Sheath fluid focuses the cell suspension, causing cells to pass through a laser beam one at a time. Forward and side-scattered light is detected, with fluorescence emitted from stained cells.

Figure 2. Overview of basic multicolor flow cytometry technology

1. When a sample is introduced into the multicolor flow cytometer flow chamber, it enters the fluidics system and separates into single cells in a process known as hydrodynamic focusing. Hydrodynamic focusing uses a controlled fluid flow to focus the sample into a narrow diameter, causing the cells to separate and align in a single file.
2. As each cell passes the laser, the instrument records it as an event. For each event, forward scatter (FS) and side scatter (SS) are subsequently recorded. If a cell is fluorescently labeled, the laser excites the fluorophore, and the emitted light is collected as fluorescence intensity.
3. For the instrument to detect the specific wavelength emitted by a fluorophore, the emitted light is passed through a series of mirrors and filters until it reaches the appropriate detector. Detectors are known as photomultiplier tubes (PMTs) and will only detect fluorescence at a specific wavelength.
4. Optical filters block certain wavelengths and let others pass. When placed at an angle, a dichroic filter acts as a mirror, allowing specific wavelengths to pass through while reflecting others. The type and order of dichroic filters allow the simultaneous detection of multiple signals.

Measurement of forward and side scattered light

All cells or particles passing through the beam scatter laser light, measured as FS by detectors in front of the light beam, and SS, measured from detectors to the side of the light beam (Figure 3).

Figure 3. Light scatter as the green laser interrogates the cell. The direction of light scattered by the cell correlates to cell size and granularity.

Cell populations are often distinguishable based on differences in their size and granularity because FS correlates with cell size, and SS is proportional to the granularity of the cells (Figure 4).

Figure 4. a) Flow cytometry measures the FS and SS of laser light, reflecting cell size and granularity. b) Typical plot distinguishing immune cell types based on FS and SS data.

A helpful example of this is running blood samples on the flow cytometer.

• Larger and more granular granulocyte cells show as a large population with a high SS and FS.

• Monocytes are large cells, but not so granular, so these produce a separate population with a high FS but lower SS.

• Smaller lymphocytes and lymphoblasts form a separate population with less FS, and also have a low SS as they are not granular cells.

Therefore, these cells can be separated into different populations based on their FS and SS alone (Figure 5).

Figure 5. Flow cytometry graph: dot plot of FS versus SS. Each dot represents a single cell analyzed by the flow cytometer. Differences in cell size and granularity determine the characteristic position of different cell populations. Image reference: Riley and Idowu, Principles and Applications of Flow Cytometry.

Measurement of scattered light and fluorescence

As well as measuring forward and side scattered light from all cells or particles in a sample, fluorescence detectors within the flow cytometer measure the fluorescence emitted from positively stained cells or particles. For example, these could be stained with a fluorescently labeled antibody against a particular protein or a fluorescent ligand that binds a specific structure such as DNA. These fluorophores emit light when excited by a laser with the corresponding excitation wavelength.

FS and SS light and fluorescence from stained cells are split into defined wavelengths and channeled by a set of filters and mirrors within the flow cytometer towards sensors known as photomultiplier tubes (PMTs). The PMTs convert the energy of a photon into an electrical signal (voltage). The fluorescent light is filtered so that each sensor will only detect fluorescence at a specified wavelength.

In the example shown in Figure 6, the fluorescein isothiocyanate (FITC) channel PMT will detect light emitted from FITC at a wavelength of approximately 519 nm. The phycoerythrin (PE) channel PMT will detect light emitted from PE at 575 nm wavelength. Each PMT will also detect any other substances present in the sample emitting light at a similar wavelength to the fluorophore it is detecting.

Figure 6. Cells stained with fluorescent antibodies pass by the laser

Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT (Figure 7). Short pass (SP) filters allow the transmission of photons below a specified wavelength, whereas long pass (LP) filters allow the transmission of photons above a specified wavelength.

Dichroic filters/mirrors (such as dichroic LP mirrors) are positioned at a 45° angle to the light beam and redirect, rather than completely blocking, light of undesired wavelengths. For example, photons above a specific wavelength are transmitted straight ahead in a long pass dichroic filter, while photons below the specific wavelength reflect at a 90° angle.

Figure 7. Band pass (BP), short pass (SP), and dichroic filters in the flow cytometer

References

1. McKinnon, K.M., Flow Cytometry: An Overview Curr Protoc Immunol 5 (1),1-11 (2018)

2. Holmberg-Thyden, S.,, Gronbaek, K.,, Gang, A.O.,, et al. A user’s guide to multicolor flow cytometry panels for comprehensive immune profiling. Analytical Biochemistry 627 (114210), (2021)

3. Maciorowski, Z.,, Chattopadhyay, P.K., Jain, P. Basic Multicolor Flow Cytometry Curr Protoc Immunol 5 (4),1-38 (2017)