Stand-alone dyes

FITC

FITC (Fluorescein isothiocyanate)

Molecular Formula: C21H11NO5S

CAS Number isomer I: 3326-32-7
CAS Number isomer II: 18861-78-4

FITC (fluorescein isothiocyanate) is a derivative of fluorescein. It is the most widely used fluorescent probe for the preparation of conjugates of molecules from molecular biology or carbohydrate polymers such as dextran1.

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Structure and physical properties
FITC exhibits an excitation maximum at λ = 495 nm and emission maximum at approximately λ = 519 nm. The color of the compound is yellow while the emitted light is green. There are two different isomers of FITC:

  • Isomer I, also referred to as fluorescein 5-isothiocyanate or 5-FITC
  • Isomer II, also referred to as fluorescein 6-isothiocyanate or 6-FITC

The variation of the excitation and emission wavelengths among the two isomers of FITC is minor. FITC-functionalized biopolymers such as FITC-dextrans exhibit similar excitation and emission 0.

Plot FITC

Figure 1. Excitation(orange) and emission (blue) spectrum of FITC isomer I. The excitation maximum is 495 nm and the emission maximum is approximately 519 nm.

Depending on the isomer, FITC contains an isothiocyanate group at either position 5 (isomer I) or position 6 (isomer 2) of the bottom benzene ring, see figure 2. Isothiocyanate easily reacts with nucleophiles such as amines under mild conditions. The compound is often used in the form of one of its two isomers or sometimes as mixture of the two.

FITC

Fig 2. Structural representation of FITC isomer I. FITC isomer I (fluorescein 5-isothiocyanate) contains an isothiocyanate group at position 5.

Storage and stability
Store at 0-8 ˚C in a tightly closed container when not in use. FITC is stable under recommended storage conditions.

Solubility
FITC is soluble in DMSO and DMF.

 

Applications

General

FITC is commonly used for labeling antibodies2 (IgG) and also in other immunological applications. It is also often used in flow cytometry, a technology frequently used by biologists to study cellular populations with high precision3.

FITC-dextran 

The labelling of dextran with fluorescein via its FITC derivative was first described in a publication in 1973 by de Belder and Granath4. Now it is a long-established method for obtaining fluorescent labelled polysaccharides. FITC-functionalized polysaccharides such as FITC-dextrans are very useful for studying permeability and transport in a large variety of cells and tissues. This includes the intestinal5, neoplastic6– and ocular7 tissue as well as in research related to brain and neural system8. FITC-dextran and other FITC-conjugated polysaccharides can also be used to study the microcirculation, which can be described as the smallest circulation of blood in the microvessels. These are present within all organ tissues. FITC-dextrans have been used in studies of leukocyte adhesion, macromolecular leakage (see figure 3) and the leakage of microcirculation during ischemia/reperfusion 9. FITC-dextrans have also been used to study the intestinal mucosal microcirculation10.

hamster cheek pouch injected with 150 kDa FITC-dextran_1

Figure 3. Hamster cheek pouch injected with 150 kDa FITC-dextran. The picture was taken 1 min after injection.

pH Indicator

FITC and other fluorescent dyes have the ability of changing color in response to pH-changes. This can be utilized for measuring pH in living cells. Changes in cellular pH can reflect a range of physiological processes, including muscle contraction, endocytosis, cell proliferation, apoptosis, and ion transport11.

Fluorescent pH-indicators can be either stand-alone-dyes, such as FITC, or dyes coupled to a macromolecule, such as FITC-dextran. The advantage of using fluorescent dextran derivatives is that the molecules can be accumulated in specific intracellular compartments12. Compared to microelectrode techniques, fluorescent pH-indicators also have greater spatial sampling capability11. Another advantage of the probes and indicators is that they don’t bind to cellular proteins12.

FITC-products from TdB Labs

  • FITC (isomer I)
  • FITC -dextran
  • FITC -lysine-dextran
  • FITC-CM -dextran
  • FITC-CM -polysucrose
  • FITC-DEAE -dextran
  • FITC-DEAE -polysucrose
  • FITC -dextran sulfate
  • FITC -hydroxyethyl starch
  • FITC -inulin
  • FITC -polysucrose
  • FITC -Q-dextran
  • FITC -trehalose
  • Fluorescein Hyaluronic Acid

Read more about all applications here.

References

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All publications

  1. Fluorescein Isothiocyanate – an overview | ScienceDirect Topics. https://www.sciencedirect.com/topics/neuroscience/fluorescein-isothiocyanate.
  2. The, T. H. & Feltkamp, T. E. W. Conjugation of fluorescein isothiocyanate to antibodies. Immunology 18, 865–873 (1970).
  3. Picot, J., Guerin, C. L., Le Van Kim, C. & Boulanger, C. M. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology 64, 109–130 (2012).
  4. de Belder, A. N. & Granath, K. Preparation and properties of fluorescein-labelled dextrans. Carbohydrate Research 30, 375–378 (1973).
  5. Aden, K. et al. Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice. Gastroenterology 156, 145-159.e19 (2019).
  6. Gerlowski, L. E. & Jain, R. K. Microvascular permeability of normal and neoplastic tissues. Microvasc. Res. 31, 288–305 (1986).
  7. Elevated cAMP opposes (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium. – Abstract – Europe PMC. https://europepmc.org/article/pmc/pmc2932488.
  8. Hultström, D., Malmgren, L., Gilstring, D. & Olsson, Y. FITC-Dextrans as tracers for macromolecular movements in the nervous system. A freeze-drying method for dextrans of various molecular sizes injected into normal animals. Acta Neuropathol. 59, 53–62 (1983).
  9. Svensjö, E. et al. Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1–CXCR1/2-pathway. Microvascular Research 83, 185–193 (2012).
  10. Schmidt, C. et al. Confocal laser endomicroscopy reliably detects sepsis-related and treatment-associated changes in intestinal mucosal microcirculation. Br J Anaesth 111, 996–1003 (2013).
  11. Han, J. & Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 110, 2709–2728 (2010).
  12. Takahashi, S. et al. Development of a Series of Practical Fluorescent Chemical Tools To Measure pH Values in Living Samples. J. Am. Chem. Soc. 140, 5925–5933 (2018).

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