Flow cytometry is a widely used, laser-based method for analyzing the expression of cell surface and intracellular molecules. Discover more with our introduction to flow cytometry.
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There are many applications of flow cytometry in research and diagnostics, including simultaneous multi-parameter analysis of single cells and characterizing and defining different cell types in heterogeneous cell populations. 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.
A flow cytometry test is usually based on measuring 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 staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated in tubes or microtiter plates with unlabeled or fluorophore-labeled antibodies and analyzed on the flow cytometer.
Multicolor flow cytometry takes this further by analyzing multiple parameters on thousands of single cells or other particles in seconds2,3.In multicolor flow cytometry, fluorescent markers 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.
If you arelooking to get to grips with flow cytometry analysis, check out our free online flow cytometry training.
- Understanding the instrumentation basics
- The flow cytometer
- The multicolor flow cytometer
- Measurement of forward and side scatteredlight
- Measurement of scattered light and fluorescence
When a cell suspension is run 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 1.4
When a cell suspension is run 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 14.
Figure 1. Flow cytometry diagram giving 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, as well as fluorescence emitted from stained cells.
Figure 2. Overview of basic multicolor flow cytometry technology
- 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.
- As each cell passes the laser, the instrument records it as an event. For each event, forward scatter and side scatter are subsequently recorded. If a cell is fluorescently labeled, the laser excites the fluorophore, and the emitted light is collected as fluorescence intensity.
- 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.
- Optical filters block certain wavelengths and let others pass. A dichroic filter acts as a mirror when placed at an angle, allowing specific wavelengths to pass through while others are reflected. The type and order of dichroic filters allow the simultaneous detection of multiple signals.
All cells or particles passing through the beam scatter laser light, measured as forward scatter (FS) by detectors in front of the light beam, and side scatter (SS), measured from detectors to the side of the light beam (Figure 3).
Figure 3. Light scatters as the green laser interrogates the cell. The direction of light scattered by the cell correlates to cell size and granularity.
FS correlates with cell size, and SS is proportional to the granularity of the cells. Therefore, cell populations can often be distinguished based on differences in their size and granularity (Figure 4).
Figure 4. a) Flow cytometry measures the forward scatter (FS) and side scatter (SS) of laser light, reflecting cell size and granularity. b) Typical plot showing how different immune cell types can be distinguished 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 are seen 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.
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 (or fluorochromes) 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, the 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 are reflected at a 90° angle.
Figure 7. Band pass (BP), short pass (SP), and dichroic filters in the flow cytometer.
Read next: Antibody staining for antigen detection in flow cytometry
- McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol 2018;120:5.1.1-5.1.11
- Holmberg-Thyden S, Gronbaek K, Gang AO, et al. A user’s guide to multicolor flow cytometry panels for comprehensive immune profiling. Analytical Biochemistry 2021;627:114210
- Maciorowski Z, Chattopadhyay PK, Jain P. Basic Multicolor Flow Cytometry. Curr Protoc Immunol 2017;117:5.4.1-5.4.38