FITC (Fluorescein 5-isothiocyanate)

Technical Specifications

Product Description & Scientific Applications

Fluorescein 5-isothiocyanate (FITC, isomer I; 5-FITC; C.I. 45015) is the amine-reactive derivative of fluorescein, carrying an isothiocyanate group on the pendant benzoate ring of the xanthene scaffold. The isothiocyanate carbon is strongly electrophilic and forms a thiourea bond on contact with nucleophiles such as primary amines, which is the basis of its use as a protein-labelling reagent. Note that the common 5- and 6- designations refer to benzoate-ring positions and are transposed relative to the systematic spiro nomenclature, in which this isomer appears as 6-isothiocyanato; CAS 3326-32-7 is the identifier that distinguishes it from the 6-isomer (18861-78-4) and the isomer mixture (27072-45-3).

Photophysics. Excitation and emission maxima lie near 495 and 519 nm, with an extinction coefficient of 68,000 M⁻¹cm⁻¹ at 494 nm — the value conventionally used to calculate degree of labelling. Emission is strongly pH-dependent: the monoanion and dianion are in equilibrium with a pKa of 6.4, and the monoanion emits with a quantum yield of 0.36 against 0.93 for the dianion, so a substantial weakly emitting fraction persists near neutral pH and signal tracks buffer composition. The dye is prone to photobleaching.

Protein and antibody conjugation. Degree of labelling does not scale usefully with dye excess. An antibody carrying five fluoresceins is 50 to 70% quenched, and an initial dye-to-antibody ratio of six gave an average of only 1.3 fluoresceins per antibody in the isolated product. Fluorescence lifetime also falls at high surface density on cells, consistent with self-quenching.

Flow cytometry and immunofluorescence. Excited at 488 nm with emission collected near 519 nm through a 530/30 filter, FITC is rated of moderate brightness and sensitive to pH changes in cytometry guideline comparisons. Alexa Fluor 488 occupies the same channel and is substantially brighter and more photostable; FITC remains in wide use for its cost, its established conjugation chemistry, and the volume of published protocols built around it.

Permeability tracers. FITC-dextran conjugates are available from 4 to 70 kDa, and the molecular weight chosen determines whether solute, ion or protein permeability is what the measurement reports. Low brain concentrations of fluorescein are measurable owing to the sensitivity of fluorescence detection and its negligible binding to plasma proteins, supporting blood-brain barrier permeability work.

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