FITC (fluorescein isothiocyanate) is widely used to label proteins, antibodies, peptides, hormones, amine-modified oligonucleotides, and other amine-containing molecules with the green fluorescent dye fluorescein. The isothiocyanate moiety (-N=C=S) on FITC reacts with amino-terminal and primary amine groups on the target biomolecule to form a covalent thiourea bond. The resulting fluorescein conjugates can be used as specific probes in several applications, including enzymatic kinetics, immunocytochemistry, immunohistochemistry, fluorescence microscopy, flow cytometry, and fluorescence in situ hybridization. FITC has an excitation maximum of 491 nm which closely matches the 488 nm spectral line found in most instruments, and a relatively broad emission spectrum with a maximum of 516 nm. ⁽¹¹⁾

Although FITC (Ex/Em= 491/516 nm) and fluorescein (Ex/Em= 498/517 nm) exhibit similar spectral profiles, they are however not equivalent. FITC is a fluorescein derivative consisting of a fluorescein core and an amine-reactive isothiocyanate moiety. The isothiocyanate group reacts with primary amines forming covalent thiourea bonds that attach fluorescein to the biomolecule. Moreover, FITC is typically supplied as a mix of isomers consisting of fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). ⁽¹⁰⁾

FITC reacts with free primary amine groups of proteins to form covalent bonds. The isothiocyanate (-N=C=S) group is reactive towards any nucleophilic site, but FITC will selectively react with N-terminal amines because these bonds are far more stable. The primary amine attacks the electrophilic carbon of the isothiocyanate group, forming a thiourea bond connecting FITC and the protein in question. Conditions such as pH must be controlled, as the isothiocyanate group technically can react with secondary amines and other nucleophiles depending on reaction conditions. ⁽⁹⁾

The degree of labeling (DOL) is a method commonly used to determine the average number of fluorophores, such as FITC, that are labeled to a protein. It is calculated by separately determining the conjugate's protein and fluorophore molar concentrations based on absorbance measurements. These concentrations are then expressed as a molar ratio in the form of label:protein. For antibodies, the ideal DOL usually falls between 2 to 10. However, a more precise value will largely depend on the properties of the label and protein. This means that for many bioconjugations, the optimal DOL must be experimentally determined, often through several small-batch labelings. Before the DOL can be determined, the following specifications must be known: ⁽⁷⁾

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Calculate the degree of labeling using the following formula:

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iFluor^® 488, Alexa Fluor^® 488, and FITC are green fluorescent dyes with nearly identical spectral profiles, extinction coefficients, and fluorescence quantum yields (see table below). Like FITC, iFluor 488^® succinimidyl ester and Alexa Fluor^® 488 NHS ester label the primary amines of proteins, antibodies, and other amine-containing molecules with their respective dyes. Because of these spectral similarities, either iFluor^® 488 or Alexa Fluor^® 488 can seamlessly replace FITC in any application without having to adjust the experimental setup.

iFluor^® 488 and Alexa Fluor^® 488, however, outshine FITC in all performance areas. Unlike FITC, which is susceptible to self-quenching, iFluor^® 488 and Alexa Fluor^® 488 can be labeled to proteins at high molar ratios with minimal self-quenching resulting in brighter, more sensitive conjugates. iFluor^® 488 and Alexa Fluor^® 488 labeled probes are highly photostable and insensitive to changes in pH, whereas FITC exhibits significant decreases in fluorescence intensity as the pH decreases from 10 to 3. iFluor^® 488 and Alexa Fluor^® 488 are trademarked products of AAT Bioquest and ThermoFisher, respectively. ⁽⁴⁾

Filters are used in fluorescence imaging and microscopy to improve image quality by enhancing or blocking specific wavelengths. These filters collect light more efficiently by blocking wavelengths that are not useful in the experiment. The excitation source light may be filtered before hitting the sample to reduce noise or focus on specific incoming wavelengths in cases where more than one fluorescent compound is being used. The emitted light is also filtered such that the detector is only analyzing the regions of concern, which makes experimentation more efficient. A commercial FITC filter set typically consists of three optical filters -an excitation filter, emission filter, and dichroic beamsplitter - optimized for detecting FITC fluorescence and other green fluorescent dyes, including iFluor^® 488. The excitation and emission filters are bandpass filters that transmit a wavelength range that corresponds only to the excitation and emission spectra of FITC. For example, a FITC filter set might have an excitation and emission filter with a wavelength range of 467-498 nm and 513-556 nm, respectively. ⁽⁶⁾

FITC is generally supplied as a lyophilized solid and must be resuspended in an organic solvent, such as ethanol, DMSO, or DMF, before labeling proteins or any other amine-containing biomolecule. To make a FITC stock solution, reconstitute the dye in high-quality, anhydrous DMSO, such that the concentration is approximately 1 mg/mL (i.e., for optimal results, this should be prepared fresh for each labeling reaction). Further dilution of the FITC stock solution into an aqueous buffer (e.g., HBSS) is required before performing conjugation. We do not recommend storing FITC in aqueous solutions as it is unstable in water. ⁽²⁾

FITC, as a lyophilized solid, should be stored refrigerated (2 °C to 8 °C), desiccated, and protected from light. At these conditions, FITC should be stable for up to 12 months. In solvents such as DMSO, FITC can be stored at -80 °C for 6 months or -20 °C for 1 month, protected from light and under nitrogen. Never store FITC in aqueous media as it is unstable in water. ⁽¹⁾

No, avoid using FITC and GFP together in the same application. FITC and GFP have nearly identical spectral profiles, making it virtually impossible to distinguish their fluorescence signals from one another due to significant cross-talk. It is best to use fluorophores with minimal spectral overlap for multicolor experiments. For example, green fluorescent dyes, such as FITC, pair well with red fluorescent dyes, such as iFluor^® 647 or allophycocyanin (APC). ⁽³⁾

Yes, FITC can be used with phycoerythrin (PE) and allophycocyanin (APC) for multicolor analysis. The caveat, however, is compensation using single-stained controls will be required during data analysis since two or more fluorophores have overlapping emission spectra. Compensation calculates how much interference a fluorophore will have in a channel that was not assigned specifically to measure it and prevents data misinterpretations by removing such artifacts. For the multicolor panel of FITC, PE, and APC, following excitation with the blue laser (488 nm), FITC emits a broad emission that spills into the PE detector. Additionally, PE shows spillover into the APC detector. The portion of the FITC spectrum detected in the PE detector, and the PE spectrum detected in the APC detector must be subtracted from the PE and APC signals using compensation. ⁽³⁾