FITC (Fluorescein isothiocyanate) was introduced by A.H. Coons in 1942 and has since become one of the most widely used fluorescent molecules in biochemical research and the modification of proteins and other biomolecules. With its reactive isothiocyanate group (-N=C=S), FITC has revolutionized the labeling of proteins, antibodies, and biomolecules. The fluorescent tags attached to proteins, antibodies, and lectins via amine groups, forming stable thiourea linkages by reacting with primary amines (eg. at lysine residues) and the amino terminus of proteins.

(Strept)avidin can be tagged with FITC to create highly fluorescent derivatives for staining techniques. The optimal modification range depends on the overall length and molecular weight (Mw) of the dextran. For low Mw dextran, such as 10 kDa, sufficient fluorescence can be achieved with 1 FITC per dextran molecule. However, as the Mw increases, higher labeling is required. For example, a 100 kDa dextran would need at least 10 times more labeling to achieve the same fluorescence intensity.

Note: Lower levels of labeling result in low luminescence, while higher levels can cause quenching effects, nonspecific binding, or protein precipitation.

Here, we will focus on FITC labeling techniques.

Key advantage of FITC in bioconjugation

FITC (Fluorescein isothiocyanate) offers several advantages that make it widely used in various fields:

1. High Absorptivity and Quantum Efficiency: FITC has high absorptivity and quantum efficiency, ensuring a large proportion of absorbed photons are converted into emitted fluorescence, making it an exceptionally effective fluorescent label.
2. Compatibility with Multiple Techniques: FITC can be used in a range of techniques, including flow cytometry, fluorescence microscopy, and immunohistochemistry, providing versatility in experimental design.
3. Stable Conjugation: The isothiocyanate group of FITC forms stable thiourea linkages with primary amines, ensuring durable and reliable labeling of biomolecules.
4. Multiplexing Capability: FITC can be used in combination with other fluorescent dyes, enabling multiplexing and the simultaneous detection of multiple targets in a single sample.
5. Rapid Reaction with Amines: FITC reacts quickly with amines, allowing for efficient labeling of proteins, antibodies, and other biomolecules.
6. High molecular absorptivity: FITC labels are favored over traditional colorimetric and radio labels due to their high molecular absorptivity. Fluorophores like FITC are bright, user-friendly, and do not require special waste handling.
7. Versatile Applications: FITC-labeled proteins, substrates, peptide hormones, and antibodies can be utilized as probes in flow cytometry, enzyme kinetics, immunocytochemistry, and detecting receptors on target cell surfaces.

Considerations when using FITC

1. Protein Purity and Concentration: Attaching a fluorescent label like FITC requires pure and highly concentrated proteins. Amine-containing molecules in the protein buffer can interfere with the reaction between FITC and the protein’s amines.
2. Hydrophobic Nature: Due to FITC’s hydrophobic nature, attaching too many FITC molecules can lead to protein aggregation or precipitation at high concentrations, potentially causing the experiment to fail.
3. Photobleaching: Fluorescein-based dyes exhibit a relatively high rate of photobleaching, meaning they can lose their fluorescence quickly when exposed to light.
4. pH Sensitivity: The fluorescent signal of FITC is sensitive to pH changes, which can affect its performance in different environments.
5. Fluorescence Quenching: When fluorescein is conjugated with biopolymers, it can experience fluorescence quenching, reducing its effectiveness.

Despite these challenges, FITC labeling is preferred over conventional methods because of its high molar absorptivity and the numerous advantages it offers in various applications.

Amine-Reactive Probes: Applications and Considerations

Amine-reactive probes are widely used to modify a variety of biomolecules, including proteins, peptides, ligands, amino sugars, and synthetic oligonucleotides. These dyes are primarily employed to create bioconjugates for applications in immunochemistry, fluorescence in situ hybridization (FISH), cell tracing, receptor labeling, and fluorescent analog cytochemistry.

In these applications, the stability of the chemical bond between the dye and the biomolecule is crucial. The bioconjugate often undergoes several post-processing steps, such as washing, permeabilization, fixation, and mounting.

Maintaining the integrity of the bioconjugate throughout these processes is essential for the fluorescence signal to accurately reflect the abundance or localization of the bioconjugate’s molecular target.

Characteristics of an Ideal Bioconjugate

The ideal bioconjugate should exhibit high fluorescence output while retaining the essential functional properties of the unlabeled biomolecule, such as:

• Solubility
• Selective binding to receptors or nucleic acids
• Activation or inhibition of specific enzymes
• Ability to integrate into biological membranes

However, it is important to note that conjugates with a high degree of labeling can sometimes precipitate out of solution or bind non-specifically.

some amine-reactive probes are vital reagents for various bioanalytical applications, including amine quantitation, protein and nucleic acid sequencing, and chromatographic and electrophoretic analysis of low molecular weight molecules. These probes play a crucial role in enhancing the accuracy and efficiency of bioanalytical techniques.

Stability and Reactivity of Isothiocyanates

Isothiocyanates are an integral part of fluorescein isothiocyanate (FITC), which is widely used in bioconjugation. Unlike isocyanates, which are highly susceptible to decomposition during storage, isothiocyanates are moderately reactive but quite stable in water and most solvents. When isothiocyanates react with amines, they form thioureas, which are reasonably stable. However, thiourea formed by the reaction of FITC with amines can be converted to guanidine by concentrated ammonia.

Despite the growing number of amine-reactive fluorophores available, fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) remain widely used reactive fluorescent dyes for preparing fluorescent bioconjugates. Their stability and effectiveness make them popular choices in various biochemical applications.

FITC Staining

FITC-labeled molecules can be used as a stain in cell analysis, providing a fluorescent marker that helps in identifying and studying various cellular components and processes.

Fluorescein-dextran

The labeling of dextran with fluorescein via its derivative fluorescein isothiocyanate (FITC) was first described by de Belder and Granath in 1973. This method has since become well-established for obtaining fluorescent-labeled polysaccharides, known as FITC derivatives. The fluorescein moiety is bound by a thiocarbamoyl linkage, which provides good stability both in vitro and in vivo (Read more).

Fluorescein can also be conjugated to large molecules to examine the integrity of cell membranes and tissue junctions. Dextran is a typical molecule used for this purpose. Fluorescein isothiocyanate-dextran has been used to examine intestinal permeability. Also, FITC-dextran has been employed to investigate the translation of a subset of mRNAs encoding secretory proteins potentiated by RanBP2/Nup358.

The movement of FITC-dextran is used to measure:

• Blood-Brain Barrier Permeability: FITC-dextran serves as a tracer to assess the permeability of the blood-brain barrier.
• Colonic Epithelial Barrier Permeability: It is used to evaluate the permeability of the colonic epithelial barrier.
• Macropinocytosis: FITC-dextran helps in studying the process of macropinocytosis.

Applications of FITC labeling

Flow Cytometry: 

In flow cytometry, FITC-labeled cells pass through a thin capillary one by one. When these cells are excited by a laser, the resulting fluorescence is measured. The number of fluorescence events, or the extinction number, is used to determine the cell count.

Receptor-Mediated Targeting 

Overexpression of specific receptors can happen in cells, especially cancer cells. Many cells, including cancer cells, overexpress specific receptors such as the folate receptor. Receptor-mediated targeting employs antibodies or receptor substrates to deliver drugs directly to these cells, thereby increasing the drug concentration in diseased tissues. FITC is frequently used to label drug carriers, enabling researchers to monitor their entry into target cells both in vitro and in vivo.

Fluorescent Probe (Labelling antibodies) 

Fluorescein-labeled antibodies can be used as histochemical stains to detect antigens within tissue cells. The antibody molecules can be conjugated with chemical compounds without disrupting their ability to react with their corresponding antigens. This makes them highly effective for precise antigen detection in various histochemical applications.

pH Indicator 

FITC is pH sensitive, and both as a free dye and as a fluorescent macromolecule (e.g., FITC-dextran), its color changes in response to pH variations. This feature can be utilized to measure pH changes inside cells during various physiological processes such as apoptosis, cell proliferation, and ion transport (Read more).

Advantages of using FITC and its macromolecule complexes:

1. 1. Versatile pH Measurement: FITC can measure pH changes inside cells, aiding in the study of physiological processes.
   2. Probing Cellular Compartments: Macromolecule complexes like FITC-dextran can probe different cellular compartments. For instance, fluorescein-labeled heparin derivatives show that internalization into cellular compartments depends on sulfation patterns.
   3. Comprehensive Analysis: These probes can enter all cellular compartments simultaneously, unlike microelectrodes, which can only analyze one portion of a cellular component at a time.
   4. Non-binding Nature: Fluorescent-based indicators and probes do not bind to intracellular proteins after conjugation, ensuring accurate measurements.

Apoptosis detection

The application of FITC in the detection of apoptosis is through:

• direct DNA labeling with fluorescein-tagged nucleotides
• conjugating fluorescein with annexin which is a family of eukaryotic proteins that can bind to phospholipids in the presence of calcium ions and therefore play essential roles in various cellular processes, such as membrane organization, vesicle trafficking, and signal transduction. In humans, annexins are involved in important physiological functions like inflammation regulation, blood coagulation, and apoptosis. Using a microscope or flow cytometry, apoptotic or dead cells can be easily detected by fluorescein-labeled annexin.

Nucleotide Labeling

FITC-tagged nucleotides are widely used in cell proliferation assays. These assays help in tracking and measuring the growth and division of cells, providing valuable insights into cellular processes and the effects of various treatments on cell proliferation.

FITC -Biotin, Avidin, Lectins

FITC can be easily conjugated with antibodies against various tags. It also enables highly targeted detection when coupled with avidin or streptavidin, as both, along with biotin, can be linked to FITC through their available amine variants. Lectins, which are proteins with a high binding affinity for carbohydrates, can be used for different purposes such as staining plasma membranes (using FITC-conjugated Triticum Vulgaris lectin) or staining vascular endothelial cells (using B4, a fluoresceinated isolectin).