Intrinsically fluorescent compounds in the cell are limited; therefore, in order to visualise different aspects of the cell anatomy, cells are stained with fluorescent dyes (probes) to allow us to see biological components that would otherwise not be visible. These probes are used to identify a wide variety of components from cell surface receptors or intracellular organelles to nucleic acid components and markers of apoptosis.

Following staining and uptake into the flow cytometer, the labelled cells pass through the interrogation point and the fluorophores are excited by incident light of appropriate wavelength. Upon return to their electronic ground state, the fluorophore releases photons that are detected by the photo detectors. By employing several fluorescent dyes with differing excitation and emission wavelengths, simultaneous measurement of multiple cell properties can be achieved ( Figure1).

Fig1. Commonly used fluorescent dyes and laser sources used for excitation.

Fluorescent Dyes

 Perhaps the most commonly used fluorescent dye, in part because it was one of the first employed for flow cytometry, is fluorescein isothiocyanate ( FITC). FITC absorbs light within the range 400–550 nm, with a maximum absorbance at 490 nm; it is often excited by an argon-ion laser delivering blue light with a wavelength of 488 nm. As this is very close to the absorption (=excitation) maximum, the fluorescence emission intensity will, consequently, be maximised. The FITC emission spectrum shows maximum intensity at a wavelength of 518 nm. The difference between the maxima of the absorption and emission spectra is known as the Stokes shift. FITC is particularly sensitive to photobleaching and a number of FITC derivatives have been developed, including Alexa Fluor ® 488, to overcome this issue.

A set of very versatile fluorescent dyes can be used to stain nucleic acids, as well as to assess membrane integrity or analyse the cell cyle. Commonly used dyes in this context include propidium iodide, 7-aminoactinomycin D (7-AAD) and 4′,6-diamidi no-2-phenylindole ( DAPI). These dyes are excited at different wavelengths and the choice of which dye to use is often dependent on the surface markers being stained in tandem with them. For example 7-AAD can be excited by blue light, whereas DAPI is excited by UV light.

Tandem dyes comprise another tool that has added to the versatility of fluorescence labels; examples include phycoerythrin-Cy7 and allophycocyanin-Cy7. The tandem dye concept employs the process of fluorescence resonance energy transfer ( FRET, whereby the fluorophore with the lower excitation wavelength is excited by the incident light and the fluorophore with the higher excitation wave length emits the fluorescence photons. Therefore, the wavelength difference between the excitation and emission wavelength is increased as compared to a single fluorophore, resulting in a larger Stokes shift.

Most recently, a new family of organic polymers (including Brilliant Violet TM (BV) 605, BV650, BV711 and others) that are excited by violet and ultraviolet light has enabled a rapid expansion of available fluorescence labels, allowing for 14–20 colours to be regularly employed on flow cytometers for research and clinical purposes.

Toxins

Additionally, fluorescently labelled toxins can also be used to look at specific features of the cell. One of the best examples of this approach is the fluorescent conjugate of phalloidin, a phallotoxin from the death cap mushroom that binds and stabilises filamentous actin ( F-actin) and allows for assessment of total F-actin by flow cytometry. Toxins typically have a dissociation constant ( K_d ) in the low nanomolar range and thus make exquisitely sensitive markers when coupled to fluorescent dyes.

Fluorescent Proteins

 Another type of molecule that can also be used in flow cytometry is fluorescent proteins. One of the most commonly used is green fluorescent protein (GFP), which, unlike the other proteins discussed below, will be expressed by the cell population of interest. GFP is excited at 489 nm and can be used in multiple applications, including sorting of GFP transfected cells and fate mapping of specific cell populations in trans genic animals.

Another two regularly employed fluorescent labels are phycoerythrin (PE) and allophycocyanin (APC). These are phycobiliproteins , water-soluble proteins that are important in capturing light energy and are present in cyanobacteria and certain types of algae. Compared to FITC labelling, use of either of the two phycobiliproteins results in a five- to tenfold greater fluorescence emission intensity. Like FITC, an argon laser can be used to excite PE. Owing to its higher absorption maximum (λ_exc = 650 nm), APC is usually excited by a helium-neon laser producing red light.

Antibodies

Monoclonal or polyclonal antibodies are typically conjugated to fluorescence dyes to enable specific labelling of cell populations. Different dyes use different conjugation chemistry; for example, FITC reacts with the amino group of lysine residues. Given that immunophenotyping is one of the most common uses of flow cytometry, a vast array of antibodies is available that are directed towards cell surface markers defining immune cell populations and their activation states, as well as intracellular factors such as cytokines and transcription factors. Surface markers in immunology are often referred to as clusters of differentiation ( CD; currently 371 for humans) while cytokines are typically referred to as interleukins (Ils). Similar to antibodies, another type of molecule that can be used to identify specific receptors are affibodies . Affi bodies imitate monoclonal anti bodies but are designed to be expressed in a bacterial expression system.

Label Selection and Experiment Design

 Emission spectra of fluorescent dyes and reporter molecules extend over a wide wave length range and hence there can be substantial overlap (spectral overlap) when several dyes are used; for example, a fraction of the FITC signal overlaps with that of PE and vice versa. Such overlap can be corrected in a process called colour compensation, whereby a fraction of the emission by one fluorophore (acquired in one channel measured for a particular sample) is subtracted from the emission by another (acquired in a different channel). When only a small number of fluorophores are involved, this process is relatively simple and can be easily performed manually. For large numbers of fluorophores to be analysed, automated methods have been established. The assessment and correction of this spectral overlap involves running single stains of each colour into the flow cytometer to establish the emission of each fluorophore individually.

When compiling panels of fluorescent dyes for an experiment to investigate multiple aspects of a cell’s characteristics, it is important to appraise the parameters of different fluorophores and their spectral overlap profile. The reduction of spectral overlap helps to minimise the risk of incorrect analysis. For example, some fluorescent dyes are brighter than others and should therefore be employed for surface markers that are expressed at lower levels on cells. Additionally, some fluorophores have a fairly high molecular mass, such as PE and APC, and are thus less useful for intracellular staining. PE and APC also tend to generate much higher background staining than some of the other fluorescent labels.

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مواضيع ذات صلة

Fluorescence-Activated Cell Sorting (FACS)

Flow Cytometry – Instrumentation

Antibody Preparation

Harnessing the Immune System for Antibody Production

Electrophoretic Techniques : Support Media and Buffers

Principles of Liquid Chromatography

Preliminary Purification Steps

Cell Disruption and Production of Initial Crude Extract

Determination of Protein Concentrations

Molecular Biotechnology and Applications

The Biology of Restriction Enzymes

The Isolation of DNA