Flow Cytometry – Types, Purpose, Reagents, Examples, Application

• Flow cytometry is a technique that allows for the quick multi-parametric study of single cells in solution.
• Flow cytometers use lasers as light sources to generate scattered and fluorescent light signals, which are read by photodiodes or photomultiplier tubes.
• These signals are translated into electronic signals, which are then processed by a computer and written to a data file in standard format (.fcs).
• Based on their fluorescent or light scattering features, cell populations can be studied and/or isolated.
• In flow cytometry, numerous fluorescent reagents are applied. In this category are fluorescently conjugated antibodies, DNA binding dyes, viability dyes, ion indicator dyes, and fluorescent expression proteins.
• Flow cytometry is a potent technique with applications in immunology, molecular biology, bacteriology, virology, cancer biology, and monitoring of infectious diseases.
• Studies of the immune system and other aspects of cell biology are now possible with a level of precision never before attained.

Flow cytometry

• Flow cytometry is a technique that analyses single cells or particles as they flow past a single or several lasers in a buffered salt solution.
• Each particle is examined for visible light scatter as well as one or more fluorescence characteristics.
• Visible light scatter is assessed in two directions, the forward direction (Forward Scatter or FSC), which might show the relative size of the cell, and at 90 degrees (Side Scatter or SSC), which indicates the internal complexity or granularity of the cell.
• Fluorescence is independent of light scattering. Transfection and production of fluorescent proteins (e.g., Green Fluorescent Protein, GFP), staining with fluorescent dyes (e.g., Propidium Iodide, DNA), or staining with fluorescently labelled antibodies are used to prepare samples for fluorescence measurement (e.g., CD3 FITC).
• Flow cytometry is a versatile technique with applications in numerous fields, including immunology, virology, molecular biology, cancer biology, and monitoring of infectious diseases.
• For instance, it is a highly successful method for researching the immune system and its response to infectious diseases and cancer.
• It permits the simultaneous analysis of mixed populations of cells from blood and bone marrow in addition to solid tissues that can be dissociated into single cells, such as lymph nodes, spleen, mucosal tissues, solid tumours, etc. In addition to analysing cell populations, one of the most important applications of flow cytometry is sorting cells for subsequent study. Later in this unit, applications will be examined in greater depth.
• Flow cytometry’s apparatus has changed during the past several decades. Multiple laser systems and specialised equipment, such as systems with 96-well loaders optimised for bead analysis, systems that combine microscopy and flow cytometry, and systems that combine mass spectrometry and flow cytometry, are commonplace.
• In recent years, the expansion in accessible reagents has led to an explosion in the number of parameters utilised in flow cytometry research.
• Fluorochromes used to conjugate monoclonal antibodies, such as tandem dyes and polymer dyes, have increased dramatically.
• In addition, the spectrum of fluorescent proteins available for transfection has expanded to include mCherry, mBanana, mOrange, mNeptune, etc.
• These advancements in fluorochromes and equipment have enabled tests with over thirty parameters.
• Data analysis is the final step of a flow cytometry experiment. Traditional histogram (dot plot) gating and analysis with two parameters is still widely employed.
• However, as the number of factors and complexity of experiments expand, novel cluster data analysis algorithms such as PCA, SPADE, and tSNE are being implemented.
• These enhanced data mining techniques enable the extraction of meaningful information from the high-dimensional flow cytometry data already available.

Purpose of flow cytometry

Flow cytometry is a well-established method for identifying cells in solution, and it is most frequently used to evaluate peripheral blood, bone marrow, and other bodily fluids. Flow cytometry is utilised to identify and quantify immune cells and to characterise haematological cancers. 1 They can quantify:

• cell size.
• cell granularity.
• total DNA.
• new synthesized. 
• DNA gene expression.
• surface receptors.
• intracellular proteins.
• transient signal.

The capacity to take these measurements in a relatively short amount of time is one of the process’s most significant features. In less than one minute, they can quantify up to three to six features or components in a single sample, cell by cell, for around 10,000 cells.

How does flow cytometry work?

The first step in beginning flow cytometry is to prepare the sample for analysis. It is necessary to suspend cells obtained from cell culture, blood, or disaggregated tissues. This cell suspension is divided among several tubes for staining, but a portion of unstained cells is retained as a control. Antibodies tagged with fluorescent probes or cellular component-staining dyes are utilised to stain the remaining samples. Before examining intracellular proteins, cells must be frozen (in a formalin buffer) and permeabilized (with a permeabilizing agent) so that antibodies and dyes can enter the cell. After antibody or dye incubation, the cells are washed and resuspended in a saline-based buffer for analysis. The sample is subsequently delivered to the flow cytometer after preparation.

Fluidics, optics, and electronics are the three primary components of a flow cytometer. In the flow cell, the centre portion of the cytometer, the sample material interacts with the excitation light and scatters light, which is then collected by the detection systems.

1. The fluidics system contains sheath fluid (a saline-based buffer or water) that is forced through the machine to direct the cell sample past the laser for individual measurement of each cell.
2. The optics are comprised of lasers, which emit light to the samples, and photomultiplier tubes (PMTs), which gather the signal scattered by the sample.
3. The electronics of the cytometer translate the observed signal into digital parameters that may be evaluated by software.

Forward scatter, side scatter and fluorescent signals

Forward scatter, side scatter, and fluorescence signals are the three principal output metrics of flow cytometry. Visible laser light reflects off each cell, revealing the cell’s general size and form.

Forward scatter

• Forward scatter (FSC) is the scattering that comes from the forward direction and reflects the size of the cells.
• The scatter is measured along the laser’s path, and each cell causes diffraction by bending light around its sides.
• Consequently, the intensity of FSC reflects the cell’s diameter.

Side scatter

• The side scatter (SSC) is the scatter measured at 90 degrees to the laser beam and is indicative of the granularity or complexity of the cell.
• Specific cellular features, such as granules and the nucleus, affect the direction of light waves that enter the cell, causing the light to be refracted by these structures.
• The more intense the SSC, the greater the refraction, and the greater the predicted granularity of the cell.

Fluorescent signals

• fluorescent signal is the third output measurement. This is the light released when fluorophores are excited by a laser.
• Upon excitation, fluorophores are molecules that can absorb and emit light. Photons from the laser light source are absorbed by the electrons of the fluorophore, elevating their energy level.
• In order to return to their ground state, these electrons emit energy as photons. During this process, the amount of energy lost to molecule interactions influences the wavelength of these photons.
• Each fluorophore has its own spectrum of emission, which may partially overlap with those of other fluorophores.
• Fluorophores can be used to mark antibodies, which can then bind to and detect specific antigens on or within a cell, or as a dye to stain cells directly, for example by binding to DNA.

The Flow Cytometric Process

• The cell suspension is entrained in the centre of a thin, swiftly moving liquid stream.
• The flow is organised so that there is a substantial distance between cells compared to their diameter.
• A vibrating mechanism causes the cell stream to fragment into separate droplets. The mechanism is modified so that the possibility of several cells per droplet is minimal.
• Just prior to breaking into droplets, the stream passes through a fluorescence measuring station where the fluorescent property of each cell of interest is detected.
• A charging ring is positioned precisely at the point where the stream separates into droplets.
• A charge is deposited on the ring based on the immediately preceding measurement of the fluorescence intensity, and the opposite charge is captured on the droplet as it breaks away from the stream.
• The charged droplets then pass through an electrostatic deflection mechanism that segregates droplets into containers according to their charge.
• In other systems, the charge is directly delivered to the stream, and the droplet that breaks off retains the same sign of charge as the stream.
• After the detachment of the droplet, the stream returns to neutral.
• A fluorescent molecule is attached to an antibody specific for a certain cell surface protein, which is then introduced to a mixture of cells.
• The subsequent phase is fluorescence, during which individual cells are watched as they pass through a laser beam.
• A positive or negative charge is assigned to droplets containing a single cell based on whether the cell contains a fluorescently-tagged antibody.
• Droplets carrying a single cell are then recognised by an electric field and directed into distinct collecting tubes based on their charge, allowing for simple separation of the fluorescent antibody-labeled cells.

Multicolor flow cytometry

• Multicolor flow cytometry is an effective method for analysing mixed cell populations, such as blood and tissue cells in human and animal samples.
• Fluorescent dyes (markers) such as fluorophore or propidium iodide are typically used to identify specific cell types. The ability to utilise numerous fluorescent markers concurrently enables the identification of multiple cell types and functional markers that further characterise each sample.
• There are instruments capable of measuring twelve or more colours. Various wavelengths of light emitted by the laser are used to measure these fluorescent dyes and markers in order to sort cells by kind.
• When utilising numerous markers, each marker is activated at a distinct wavelength of light to separate them.
• Adapting a standard staining panel from 4 to 6 colours to more than 12 colours is not a matter of “plug and play”; rather, it requires a methodical approach to establish appropriate parameters in a staining panel.
• According to pre-use study, the fundamental principles of panel design are most effective. In other words, preparation is essential from the onset of referencing the stain index to properly match fluorochromes based on brightness.

Types of Flow Cytometer

1. Traditional Flow Cytometers

• Traditional flow cytometers consist of fluidics, optical, and electronics technologies.
• The fluidics system comprises of sheath fluid (often a buffered saline solution) that is pressured to transport and focus the sample at the laser intercept or interrogation point, where it is examined.
• The optical system is comprised of excitation optics (lasers) and collecting optics (photomultiplier tubes or PMTs and photodiodes) that generate the visible and fluorescent light signals needed for sample analysis.
• A series of dichroic filters direct fluorescent light to specific detectors, while bandpass filters define the wavelengths of light that are measured, allowing for the detection and measurement of each distinct fluorochrome.
• Dichroic filters are filters that pass through light with shorter or longer wavelengths while reflecting the remaining light at an angle. A 450 Dichroic Long Pass (DLP) filter, for instance, allows light with wavelengths longer than 450 nm to pass through while reflecting shorter wavelengths at an angle to be directed to another detector.
• Bandpass filters detect a narrow band of light with a certain wavelength. A 450/50 bandpass filter, for instance, allows fluorescent light with a wavelength of 450 nm +/- 25 nm to pass through the filter and be read by the detector.
• The electronic system turns the signals from the detectors into computer-readable digital signals.
• Multiple laser systems are prevalent, with instruments frequently containing twenty parameters (FSC, SSC and 18 fluorescent detectors).
• New instrument systems with five or more lasers and 30–50 parameters are being introduced, however they are uncommon. Traditional flow cytometers often employ lasers with wavelengths of 488 nm (blue), 405 nm (violet), 532 nm (green), 552 nm (green), 561 nm (green-yellow), 640 nm (red), and 355 nm (ultraviolet).
• There are available additional laser wavelengths for specialised purposes. In an effort to increase sensitivity, certain devices have replaced PMTs with avalanche photodiodes (APD) for fluorescence detection.

2. Acoustic Focusing Cytometers

• This cytometer utilises ultrasonic waves to more precisely concentrate cells for laser examination.
• This sort of acoustic focusing permits greater sample input and reduces sample clogging.
• This cytometer is equipped with up to four lasers and fourteen fluorescence channels.

3. Cell Sorters

• The cell sorter is a form of conventional flow cytometer that may purify and collect samples for further examination.
• A cell sorter enables the operator to choose (gate) a population of cells or particles that are positive (or negative) for the specified parameters and then guide those cells to a collection vessel.
• By oscillating the sample stream of liquid at a high frequency to generate drops, the cell sorter separates cells.
• The drops are then given a positive or negative charge and sent to a certain collection vessel based on their charge by passing through metal deflection plates.
• Tubes, slides, and plates are acceptable collection vessels (96-well or 384-well are common).
• There are two types of cell sorters distinguished by the location of the laser interrogation point: quartz cuvette and “jet-in-air.”
• The quartz cuvette cell sorters are easy to arrange for a sort because their laser alignment is fixed.
• The “jet in air” cell sorters require daily laser alignment and are more complex to set up, but they are more versatile for detecting minute particles.

4. Imaging Cytometers

• Combining conventional flow cytometry with fluorescent microscopy, imaging flow cytometers (IFC) combine flow cytometry and microscopy.
• This enables quick single-cell and population-level morphology and multi-parameter fluorescence analysis of a sample.
• IFC can detect protein distributions within individual cells, similar to a confocal or fluorescence microscope, and can also process huge numbers of cells, similar to a flow cytometer.
• They are particularly effective in numerous applications, including cell signalling, co-localization investigations, cell-to-cell contacts, DNA damage and repair, and any application that requires the coordination of cellular location and fluorescence expression on large populations of cells.

5. Mass Cytometers

• Mass cytometers are instruments that combine time-of-flight mass spectrometry and flow cytometry.
• Instead of fluorescently-tagged antibodies, cells are labelled with heavy metal ion-tagged antibodies (often from the lanthanide series) and detected using time-of-flight mass spectrometry.
• Mass cytometers lack FSC and SSC light detection, making the standard method of detecting cell aggregates impossible. Nevertheless, other techniques, such as cell barcoding, can be used for this purpose.
• Also, mass cytometry lacks autofluorescence signals from cells, and reagents lack the emission spectrum overlap associated with fluorescent labelling, therefore correction is unnecessary.
• However, because the sample is destroyed during analysis, cell sorting is not possible, and the acquisition rate is significantly lower than that of a typical flow cytometer (1,000 cells/second as opposed to 10,000 cells/second).
• The number of commercially available reagents for 40 channels will rise with the introduction of other metal ions, such as platinum, for conjugation with antibodies.

6. Cytometers for Bead Array Analysis

• Multiplex bead arrays for assessing huge quantities of analytes in tiny sample volumes have gained popularity.
• Briefly, these assays use capture beads with a defined quantity of fluorescence in a given channel and a reporter molecule detected by a separate laser to quantify the amount of analyte caught by a single bead.
• It is roughly similar to one hundred ELISA tests.
• To assess these tests, compact flow cytometers with typically two lasers and 96-well loaders have been created.
• These instruments have tiny footprints and optical bench designs that are tuned for detecting and distinguishing beads with varying fluorescence levels along two channels.
• There have been created instruments that can identify 100–500 distinct bead combinations.

7. Spectral Analyzers

• Compensating (or removing spectral overlap) between flurochromes is one of the difficulties of multiparameter flow cytometry. The spectral analyzer is a novel type of flow cytometer built expressly to overcome this issue.
• A spectral analyzer examines the complete emission spectrum of each fluorochrome in a multicolor sample to produce a spectral fingerprint.
• Then, each spectrum is separated during analysis to provide a pure signal for each fluorochrome.
• As a detection method for high-dimensional flow cytometry, spectral analysis is gradually replacing standard PMTs.

8. New Detector Technologies

• Photomultiplier tubes (PMTs) continue to be the detector technology of choice for flow cytometry. They are useful for fluorescence technology due to their great sensitivity and low background.
• However, certain cytometers are beginning to use solid-state detectors. Avalanche photodiodes (APDs) are affordable, sensitive, and extremely linear, and their spectrum responsiveness is enhanced in the long red region.
• Silicon photodiodes (SiPDs) are another potential solid state detector technology.

Reagents Used in Flow cytometry

Small Organic Molecules

• Small organic molecules such as fluoroscein (MW=389 D), Alexa Fluor 488 (fluorescein analog), Texas Red (325 D), Alexa Fluor 647 (1464 D), Pacific Blue and Cy5 (762 D) are commonly used for antibody conjugation.
• Their emission spectra are constant, however they have a slight Stokes shift (the difference between excitation wavelength and emission wavelength, approximately 50–100 nm).
• Additionally, they are stable and reasonably simple to conjugate with antibodies. The Alexa Fluor (Thermo Fisher) dyes were engineered to be more resistant to photobleaching, making them superior reagent options for samples that will also be utilised for imaging.

Phycobiliproteins

• Large protein molecules generated from cyanobacteria, dinoflagellates, and algae compose phycobiliproteins.
• For instance, phycoerythrin (PE) has a molecular weight of 240,000 daltons.
• Large Stokes shifts (75–200 nm) and steady emission spectra characterise these proteins.
• Due to their enormous size, phycobiliproteins are ideal for quantitative flow cytometry, as their ratio of protein to fluorochrome following conjugation is typically 1:1.
• However, phycobiliproteins are vulnerable to photobleaching and should not be used in applications involving prolonged or repetitive excitation source exposure.
• Phycoerythrin (PE), allophycocyanin (APC), and peridinin chlorophyll protein are phycobilibrotein examples (PerCP).

Quantum Dots

• Quantum Dots (Qdots) are semiconductor nanocrystals with fluorescence emission spectra that are proportional to the nanocrystal’s size.
• They are optimally excited by ultraviolet or violet lasers, whereas numerous lasers can only minimally excite them.
• This low excitation hampers fluorescence correction in multi-parameter investigations with Qdots.
• Due to compensating difficulties and the difficulty of conjugating Qdots to antibodies, polymer dyes have essentially supplanted these reagents in multiparameter staining panels.

Polymer Dyes

• Based on the length of the polymer chain and the molecular subunits that are linked, polymer dyes can be “tuned” to absorb and emit light at certain wavelengths.
• The quantum efficiency of these dyes is comparable to phycobiliproteins, but their photostability is much enhanced.
• Since polymer dyes can be engineered to absorb light selectively at specified wavelengths, they avoid the challenges with repeated laser excitation that make it problematic to employ Qdot reagents in multiparameter investigations.
• Brilliant Violet (BV), Brilliant Ultraviolet (BUV), and Brilliant Blue (BB) are examples of these reagents.

Tandem Dyes

• Tandem dyes chemically combine phycobiliproteins (PE, APC, PerCP) or polymeric dyes (BV421, BUV395) with tiny organic fluorochromes (Cy3, Cy5, Cy7) to produce a dye that use fluorescence energy transfer (FRET) to enhance the number of fluorochromes that can be activated with a single laser source.
• For instance, Texas Red has a maximum excitation wavelength of 589 nm, while PE has an emission wavelength of 585 nm; therefore, by connecting PE to Texas Red, the emission from PE is used to excite Texas Red via FRET, allowing PE-TxRed to be activated by a 488 nm or 532 nm laser.
• The polymer chain antibodies increase the number of fluorochromes that can be stimulated by a single laser using the same technique.
• The high Stokes shift values (150–300 nm) of tandem dyes are advantageous when dealing with low antigen density.
• However, tandem dyes are less stable than donor fluorochromes and their energy transfer efficiency might vary from lot to lot, which complicates correction. The majority of longer Brilliant polymer dyes are tandems and share similar problems.

Metal Conjugates for Mass Cytometry

• Antibodies utilised in mass cytometry are attached to single isotope lanthanide series heavy metal ions.
• There are now 35 commercially available lanthanide series isotopes for antibody conjugation.
• Non-fluorescent and solely suited for mass cytometry, these probes are non-fluorescent. As soon as other metal elements are assessed for compatibility with this technology, more antibody conjugates will become available.

Fluorescent Proteins

• Fluorescent proteins are extensively employed as gene expression reporter systems. Green fluorescent protein (GFP) generated from the jellyfish Aequorea victoria is the most common.
• The GFP gene was cloned to produce cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (YFP). From mushroom anemone, red fluorescent protein (DsRed) was isolated and cloned for use in protein expression systems.
• Cloned from DsRed, the monomeric fluorescent proteins of the next generation (mCherry, mBanana) have larger excitation and emission spectra.
• In flow cytometry, the violet and green/yellow excited fluorescent proteins are utilised extensively.
• Several hundred fluorescent proteins with excitation and emission spectra ranging from the ultraviolet to the near-infrared are now known and being continuously discovered and synthesised.
• The advent of several laser wavelengths on contemporary flow cytometers has vastly increased the usage of fluorescent proteins in flow cytometry.

Nucleic Acid Dyes

• Nucleic acid dyes can bind both DNA and RNA. They are utilised to quantify DNA for cell cycle analysis (Propidium Iodide, 7AAD, DyeCycle Violet, DAPI), distinguish chromosomes for sorting (Hoescht 33342, Chromomycin A3), sort stem cells via side population analysis (Hoescht 33342), and sort microorganisms.
• To determine proliferation, they can be coupled with another marker, such as fluorochrome-conjugated anti-BrdU.

Proliferation Dyes

• By pulsing cells with BrdU (bromodeoxyuridine) and then staining them with an antibody against BrdU and a DNA dye, cell proliferation can be evaluated.
• However, this method does not permit long-term research of proliferation. Multiple divisions of proliferating cells can be tracked using Carboxyfluoroscein succinimidyl ester (CFSE) and other related dyes.
• Excited red and violet variations of these dyes are now also available. Each cell is permanently branded with the dye, and succeeding generations of cells inherit less dye due to the dye’s diluting effect.
• These dyes have no effect on cell growth or shape, making them excellent for long-term studies of cell proliferation.

Viability Dyes

• The integrity of the cell membrane can be determined through the exclusion of dyes (Propidium iodide, DAPI) or the binding of a dye to amines within the cell.
• The exclusion dyes cannot be fixed and are only appropriate for non-infectious cells that will be evaluated immediately.
• Amine binding dyes, such as the Live/Dead reagents (ThermoFisher), Zombie dyes (Biolegend), or Fixable Viablity dyes (BD Biosciences), can be used to fix and stain infectious cells, cells that must be stained for internal antigens, and cells that must be preserved prior to acquisition.

Calcium Indicator Dyes

• Calcium indicator dyes exhibit a hue change upon calcium binding.
• They serve as indicators of cellular activity and signalling.
• The data is expressed as a ratio of the two wavelengths corresponding to calcium bound and unbound and dye.
• The most prevalent dye remains indo-1, a biphasic UV calcium probe. Also accessible are blue-green calcium probes, including fluo-3.

Flow cytometry data analyzed

Histograms and dot plots are generated from the gathered flow cytometry data to facilitate analysis. But first, a number of processing steps are required to permit reliable analysis.

Selection of viable single cells

• Typically, an analysis begins with the selection of single cells of interest based on specific features, a process called as gating.
• Users are able to draw lines, known as gates, around specific cell populations to choose them for further examination using analysis software.
• It is necessary to gate for single cells because doublets, cells that cross the laser at the same time, can result in false-positive signals from one of the cells.
• Using dot plots displaying FSC and SSC data, isolated cells with the size and granularity of an interest population are chosen for further research.
• Additionally, it is crucial to monitor for viability, as dead cells have a tendency to release more autofluorescence, which can interfere with analysis.
• Some of the employed fluorophores may overlap with autofluorescence, resulting in false-positive readings.
• Using viability dyes, which can bind to DNA or react with free amine groups on and within the cells, viability is determined.

Compensation

• The subsequent phase in the study is the evaluation of the fluorescent signals measured for each cell. As the emission spectra of accessible fluorophores overlap, investigations involving numerous fluorophores may necessitate correction.
• The detected signal may originate from another fluorophore if overlapping spectra produce a false-positive result. Constructing fluorophore panels with low spectrum overlap necessitates an appropriate experimental design.
• Even then, overlap correction or compensation is frequently required. A positive and negative control sample directs the compensation, ideally using the examined cell type or, if not possible, compensation beads. Compensation can be set either during the measurement or during the subsequent analysis.

Gating and analysis

• Once compensation has been established, each marker can be examined by collecting information from each fluorescence detection channel.
• On populations of interest, additional gating can be applied to zero in on the presence or lack of particular parameters.
• For this purpose, both density and histogram charts might be utilised. Individual cells are represented as dots scattered according to their fluorescence properties on density graphs.
• Colors are used to represent the number of cells in the study that possess a given attribute. The simultaneous visualisation of two markers, one on the X-axis and the other on the Y-axis, allows for the differentiation of cells based on the expression of these two characteristics.
• Histograms depict a single parameter and indicate cell counts on the Y-axis and fluorescence intensity on the X-axis.
• Proper gating requires controls, which can be established depending on positive and negative controls.
• A positive control is a cell sample that expresses the marker recognised by an antibody conjugated to the fluorophore of interest.
• A negative control may consist of unstained cells or cells stained with an isotype control, which is an antibody labelled with the fluorophore used in the study and directed against an antigen not present on the cell, to adjust for non-specific background.
• For larger panels with more characteristics, fluorescence-minus-one (FMO) controls are recommended. These controls consist of all fluorescent markers on a single flow panel, minus one, and must be constructed for each fluorescent marker used in the experiment. It enables the detection of background signals from spectral profiles that overlap.

Analysis of high-dimensional data

• The rise of the number of evaluated parameters has altered the analysis of flow cytometry data. With each additional parameter, the data acquires additional dimensions.
• In order to extract information from these high-dimensional data, new analysis techniques have been created. Principal component analysis (PCA), spanning-tree progression analysis of density-normalized events (SPADE), and t-stochastic neighbour embedding (tSNE) are examples of such approaches.
• These methods enable two-dimensional analysis by clustering data based on phenotypic similarities and differences across cells.

FACS Sorting/facs flow cytometry

• Flow cytometry is a specialised kind of fluorescence-activated cell sorting (FACS).
• It provides a method for sorting a heterogeneous mixture of living cells into two or more containers based on the individual light scattering and fluorescence features of each cell.
• It differs from flow cytometry in that it gives distinctive characterisation rather than simply counting and sorting cells.
• It is usual for the two concepts to collaborate in a co-characterization process to provide a comprehensive qualitative and quantitative method for flow cytometric analysis.

Challenges and limitations of flow cytometry

• Despite the benefits that flow cytometry can provide to cell biology and therapeutic applications, the technology has several disadvantages.
• Fluid dynamics in the system can be difficult to manage, as cells or debris can cause blockages or variations in flow rates that affect analyses.
• This is especially likely when analysing bigger cells, such as cancer cells. Pipeline contamination can cause the system to get clogged and flow to be disrupted.
• In order to achieve consistent results, laser alignment is also vital and should be examined frequently.
• Inability of single cell analysis to offer information on tissue features is another limitation.
• The expensive cost of cytometers and their operation, especially for high-parameter devices, can limit their application.

Applications of Flow cytometry

Flow cytometry has an abundance of techniques and applications that are applicable to a variety of academic disciplines. In this section, applications are largely categorised by field of research; however, any of these methods can be used to any field of study.

Flow cytometry applications in Immunology

• Immunophenotyping.
• Antigen Specific Responses.
• Intracellular Cytokine Analysis.
• Proliferation Analysis.
• Apoptosis Analysis 

Molecular Biology

• Fluorescent Protein Analysis.
• Cell Cycle Analysis.
• Signal Transduction Flow Cytometry.
• RNA Flow Cytometry.
• Cell Sorting.

Other Applications

• Absolute Cell Counting.
• Quantitative Flow Cytometry.
• Multiplexed Bead Array Assays.
• Phagocytosis Assays.
• Small Particle Analysis and Sorting.

Examples of Flow Cytometry Experiments

Immunophenotyping by Using Flow Cytometry

• Immunophenotyping is the most common flow cytometry application. It employs the unique capability of flow cytometry to examine mixed cell populations for many parameters concurrently.
• In its most basic form, an immunophenotyping assay consists of cells labelled with fluorochrome-conjugated antibodies that target cell surface antigens.
• Human Leukocyte Differentiation Workshops provide “cluster of differentiation” (CD) numbers to the majority of these antigens so that an uniform nomenclature may be used to identify monoclonal antibodies that target specific cellular antigens. CD3 is “cluster of differentiation number 3” and is used to designate the T cell co-receptor found on all T cells.
• The majority of immune cells have CD markers that distinguish them as a population of cells. These cell markers are referred to as lineage markers and are utilised to identify distinct cell types for further examination in every immunophenotyping assay.
• T cell markers (CD3, CD4, CD8), B cell markers (CD19, CD20), monocyte markers (CD14, CD11b), and NK cell markers (CD3, CD4, CD8) are some examples (CD56, CD161).
• In addition to lineage markers, other markers are employed to characterise each cell group. These may include activation markers (CD69, CD25, CD62L), memory markers (CD45RO, CD27), tissue-homing markers (α4/β7), and chemokine receptor markers (CCR7, CCR5, CXCR4, CCR6).
• Typically, immunophenotyping tests include intracellular markers such as FoxP3 (which defines Treg cells), cytokines (IFN-γ, TNF-α, IL-2 define TH1 cells), proliferation markers (Ki67, CFSE), and antigen-specific markers (major histocompatibility or MHC Tetramers).
• Although current instruments and reagents are capable of 28-color immunophenotyping tests, 12–15-color experiments are more typical.

Antigen Specific Responses by Using Flow Cytometry

• By stimulating cells with a specific antigen and then examining for cytokine production, proliferation, activation, memory, or antigen recognition through MHC multimers, it is possible to evaluate antigen-specific responses.
• MHC multimers are biotinylated MHC monomers (MHC-I or MHC-II) attached to a fluorescent streptavidin backbone in groups of four (tetramer), five (pentamer), or ten (octamer) (dextramer).
• These MHC multimers are “loaded” with the desired antigen and then utilised to bind to T cells that identify the antigen, so signalling the level of response to a particular antigen.
• This application is frequently employed in vaccine research.

Intracellular Cytokine Analysis by Using Flow Cytometry

• In order to do intracellular cytokine analysis, cells are treated for 2–12 hours with a protein transport inhibitor (Brefeldin A or Monensin) so that any cytokines produced by the cells can accumulate within the cell, allowing for improved detection.
• During this incubation, cells can be stimulated with various antigens, such as peptides from a vaccination, to test immunological response.
• Following treatment with a protein transport inhibitor, cells are stained for cell surface and viability markers, then frozen and permeabilized for intracellular staining with anti-cytokine antibodies.

Apoptosis Analysis by Using Flow Cytometry

• Apoptosis, or programmed cell death, is an often-studied process in immunology and other disciplines.
• It is utilised to preserve the immune system’s homeostasis by eliminating cells without provoking an inflammatory response (necrosis).
• It is the death process for clonally enlarged T cells following an immune response, self-targeting T cells, autoreactive B cells, and numerous other immune cells.
• Flow cytometry utilises numerous targets along the cascade of events associated with apoptosis to detect apoptosis.
• Annexin V staining targets the translocation of the plasma membrane, the TUNEL (TdT dUTP Nick End Labeling) assay targets the endonuclease digestion of DNA, the activation of Caspases can be targeted by antibodies and dyes, mitochondrial apoptosis is targeted by dyes that determine mitochondrial membrane potential, and chromatin condensation in the nucleus is detected by staining with Hoescht 33342.
• Annexin V is a phospholipid-binding protein that attaches to phosphatidylserine during apoptosis, when it is translocated to the outer layer of the cell membrane.
• Annexin V should be stained with a viability exclusion dye (such as propidium iodide) to confirm that the binding occurs on the outer surface of the cellular membrane.
• TUNEL is a method that uses the ability of terminal deoxynucleotidyl transferase (TdT) to identify DNA breaks associated with apoptosis with dUTP (deoxyuridine triphosphate) or BrdU.
• Before data collection, the dUTP or BrdU is labelled with a fluorophore for detection, and the cells are counterstained with a DNA dye.
• In most cases of apoptosis, the caspase signalling pathway is active. Utilizing intracellular labelling and antibodies specific to the active form of caspase 3, this is targeted.
• Additional tests employ fluorogenic substrates that, when exposed to caspase activity, are cleaved and emit light.
• Mitochondrial apoptosis does not always utilise the caspase route; hence, many detection approaches are utilised.
• The majority of these techniques examine mitochondrial membrane potential, including the use of the dye JC-1. However, there is an antibody targeting APO2.7 that is only expressed during apoptosis and is localised on the mitochondrial membrane.

Fluorescent Protein Analysis by Using Flow Cytometry

• Fluorescent proteins (GFP, mCherry, YFP, etc.) are employed as protein expression indicators.
• Typically, cells are transfected with a plasmid that encodes a gene of interest and a fluorescent protein, as well as a promotor sequence. The fluorescent protein’s expression serves as an indication of the gene of interest.
• In recent years, the development of a split bi- or tri-party fluorescence complementation connected to other proteins has made it possible to identify RNA–protein and protein–protein interactions.
• These techniques have improved the detection and separation of cells in which fluorescence is detectable only in response to a surrogate.
• This method has numerous uses, including in vivo tracking of transplanted cells, detection of bacterial or viral infections, and gene deletion in cells to further elucidate gene function.

Cell Cycle Analysis by Using Flow Cytometry

• Assays for cell cycle analysis involve the saturation labelling of DNA with a DNA-binding dye. In the majority of instances, the cells are fixed using a 70% ethanol solution, which permeates the cells, and then stained with the dye (PI, 7AAD, DAPI).
• Hoescht 33342 is one such dye that may enter living cells and stain DNA without harming the cells.
• In this sort of study, samples are obtained at a low flow rate using linear amplification, and the cell cycle phases are determined using ploidy modelling software.

Absolute Cell Counting by Using Flow Cytometry

• Every immunophenotyping investigation can use absolute cell counts. Fluorescent beads of a known concentration are acquired with the sample for the operation.
• The sample is evaluated, and the number of gated cells for the population of interest is compared to the number of beads obtained in the same sample to determine the number of cells per millilitre.

Multiplexed Bead Array Assays by Using Flow Cytometry

• Assays utilising multiplexed bead arrays consist of arrays of beads coated with antibodies against particular soluble proteins or nucleic acids.
• Each bead has a known amount of fluorescence and a distinct target, which determines its placement in the matrix.
• Up to 100 beads are incubated with the material of interest, treated with a fluorescence reporter, and then acquired on a flow cytometer equipped with at least two lasers to detect the two distinct fluorochromes.
• Utilizing fluorescence, specialised software is employed to calculate analyte concentrations.

Phagocytosis Assays by Using Flow Cytometry

• Using fluorescently tagged bioparticles or bacteria, flow cytometry can be used to detect phagocytosis.
• The bacteria are labelled with a pH-sensitive dye that fluoresces only when exposed to the acidic pH of a phagosome, suggesting that they have been phagocytosed.

Small Particle Analysis and Sorting by Using Flow Cytometry

• Enhanced sensitivity flow cytometers make it possible to detect and sort exosomes and other submicron particles.
• The analysis of exosomes, viruses, and other subcellular particles generates new applications in numerous domains, including cancer biology, cancer therapy, and vaccine creation.
• This application is still in its infancy, but techniques and instrumentation are advancing rapidly to make it more accessible in the near future.

FAQ

What is flow cytometry?

Flow cytometry is a technology used to evaluate cells for multiple reasons, including cell counting, phenotyping, cell cycle analysis, and viability. Light generated by lasers in a flow cytometer is scattered by cells in the sample, recorded by detectors, and converted into signals that can be examined and evaluated.

What are Multiplexed Bead Array Assays?

Assays utilising multiplexed bead arrays consist of arrays of beads coated with antibodies against particular soluble proteins or nucleic acids. Each bead has a known amount of fluorescence and a distinct target, which determines its placement in the matrix. Up to 100 beads are incubated with the material of interest, treated with a fluorescence reporter, and then acquired on a flow cytometer equipped with at least two lasers to detect the two distinct fluorochromes. Utilizing fluorescence, specialised software is employed to calculate analyte concentrations.

What is Quantitative Flow Cytometry?

Using a bead-based standard, quantitative flow cytometry generates a staining curve of known fluorescence amounts. The cells are then acquired using the same instrument parameters, and linear regression analysis is applied to determine the quantity of fluorescence on the cells. This can be expressed as Antibodies Bound per Cell (ABC), Antibody Binding Capacity (ABC), or Molecules of Equivalent Soluble Fluorochrome, depending on the bead system utilised (MESF). PE is the optimal fluorochrome for this application because, due to its size, it virtually always binds to an antibody with a ratio of 1:1 fluorochrome to protein. By constructing a standard curve and regression using MESF-bead data in every particular experiment, Molecular Equivalent of Soluble Fluorescence (MESF) standards can be utilised to convert arbitrary fluorescence intensity values to the number of fluorescent molecules on a cell.

How Cell sorting done in the flow cytometer?

To separate and purify cells or particles for further study, cell sorting employs a flow cytometer with cell sorting capabilities. Essentially, a cell sorter can separate any cell or particle that can be made fluorescent. It is possible to sort cells into 96 or 384 well plates, tubes, and slides. Transfected cells expressing a fluorescent protein, stem cells, tumor-infiltrating lymphocytes, tumour cells, and white blood cell populations are typical sample types. Scaling up the amount of antibody required to stain large quantities of cells is a crucial aspect of any cell sorting procedure.

What is Signal Transduction Flow Cytometry?

This application employs antibodies designed to bind to unphosphorylated and phosphorylated signalling molecules. The use of these reagents and specialized buffers in staining panels allows for the study of signaling pathways in mixed populations of cells.

What is RNA Flow Cytometry?

RNA flow cytometry combines flow cytometry with fluorescent in situ hybridization (FISH) in order to detect both RNA and protein expression. This method necessitates optimization of the staining panel, as not all fluorochrome-conjugated antibodies can resist successive 1-hour incubations at 40°C. When no antibodies are available for a target, RNA expression is a useful alternative.

References

• McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018 Feb 21;120:5.1.1-5.1.11. doi: 10.1002/cpim.40. PMID: 29512141; PMCID: PMC5939936.
• Brehm-Stecher, B. F. (2014). Flow Cytometry. Encyclopedia of Food Microbiology, 943–953. doi:10.1016/b978-0-12-384730-0.00127-0 
• Chantzoura, E., & Kaji, K. (2017). Flow Cytometry. Basic Science Methods for Clinical Researchers, 173–189. doi:10.1016/b978-0-12-803077-6.00010-2
• Berny-Lang, M. A., Frelinger, A. L., Barnard, M. R., & Michelson, A. D. (2013). Flow Cytometry. Platelets, 581–602. doi:10.1016/b978-0-12-387837-3.00029-8 
• Roederer, M., Parks, D. R., Herzenberg, L. A., & Herzenberg, L. A. (1998). Flow Cytometry. Encyclopedia of Immunology, 932–943. doi:10.1006/rwei.1999.0243 
• https://www.bu.edu/flow-cytometry/files/2010/10/BD-Flow-Cytom-Learning-Guide.pdf
• https://www.slideshare.net/tashagarwal/flow-cytometry-46618943
• https://www.abcam.com/protocols/introduction-to-flow-cytometry
• https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle
• https://www.technologynetworks.com/cell-science/articles/what-is-flow-cytometry-343977
• https://www.thermofisher.com/in/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/flow-cytometry-basics/flow-cytometry-fundamentals/how-flow-cytometer-works.html
• https://nanocellect.com/blog/how-does-flow-cytometry-work/
• https://en.wikipedia.org/wiki/Flow_cytometry
• https://my.clevelandclinic.org/health/diagnostics/22086-flow-cytometry