Fluorescein-5-isothiocyanate FITC

Other Names Fluorescein, 5-isothiocyanato- 5-Isothiocyanatofluorescein FITC isomer I Fluorescein 5-isothiocyanate Fluorescein isothiocyanate isomer 1 Fluorescein isothiocyanate isomer I Fluoroscein-5-isothiocyanate 

CA Index Name Spiro[isobenzofuran-l(3H),9 -[9H]xanlhen]-3-one, 3, 6 -dihydroxy-5-isothiocyanato-CAS Registry Number 3326-32-7 Merck Index Number 4089 Chemical Structure 

Chemical/Dye Class Fluorescent, Xanthene Molecular Formula C21H11NO5S Molecular Weight 389.38 pH Range 6.0-7.2 

Color Change at pH Weak green fluorescence (6.0) to strong green fluorescence (7.2) 

Solubility Insoluble in water soluble in ethanol, acetone, N,N-dimethylformamide UV-Visible (X ) 490 nm Melting Point 360°C 

---

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

Among the commercially available fluorescein derivatives that have been widely used are fluorescein isothiocyanate (FITC) 49, carboxyfluorescein (FAM) succini-midyl ester 50, and fluorescein dichlorotriazine (DTAF) 51. FITCs are the most commonly used fluorescein derivatives. They have been used to react with sulfhy-dryl [127], targeting reduced cysteine chains and especially amino groups in peptides or proteins [128]. 

Fig. 20.1. Confocal images of whole mounts of the ovijector region of A suum stained with phalloidin-tetramethylrhodamine isothiocyanate (TRITC) to show muscle and with an anti-RFamide antiserum coupled to fluorescein isothiocyanate (FITC) to show FaRPergic nerves. (A) Main ventral nerve cord encircles opening of ovijector where it meets the body wall and is immunopositive for FaRPs. (B) Flat-fixed preparation of the ovijector showing circular muscles and tracts of parallel FaRPergic nerves (arrows). (C) Detail of the circular muscle of ovijector and associated nerves (arrows). (D) A FaRPergic cell body is localized in the ventral nerve cord at junction with ovijector and provides innervation to ovijector muscle.

To be used as delivery carriers for biomolecules, first it is essential to check whether the conjugates can effectively enter cells. Cellular uptake experiments were performed by using fluorescein isothiocyanate (FITC) conjugated LDH as a probe. Cells (5 x 105/ 1 ml) were incubated with LDH-FITC and its uptake was measured by flow cytometry. As shown in Figure 13.4, the cellular uptake was time and concentration dependent and... 

Fluorescein isothiocyanate (FITC) is one of the most popular fluorescent probes ever created. An isothiocyanate derivative of fluorescein is synthesized by modification of its lower ring at... 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

An interesting application of CZE was the study of the interaction of human serum transferrin and fluorescein isothiocyanate (FITC). Measurements were carried out in an uncoated fused-silica capillary (total length 59 cm 75 pm i.d. effective lengths for... 

Hoogstraate AJ, Cullander C, Nagelkerke JF, Senel S, Verhoef JC, Junginger HE, Bodde HE (1994) Diffusion rates and transport pathways of fluorescein isothiocyanate (FITC)-labeled model compounds through buccal epithelium. Pharm Res 11 83-89... 

Trypan blue (0.4%), a substance that is usually applied to identify dead cells, can be used as a quencher for some of the external dextran conjugates [fluorescein isothiocyanate (FITC)-dextran] (72,129). 

Fluorescein isothiocyanate (FITC) or Lissamine rhodamine B sulfonyl chloride (RBSC). 

The pioneering immunofluorescence studies of Albert Coons and colleagues in the 1940s and 1950s (reviewed in ref. 11) established the effectiveness of fluorescein for immunofluorescence microscopy. The green emission of fluorescein isothiocyanate (FITC) was shown to provide a strong signal, well separated from blue cellular autofluorescence. Continued wide use of fluorescein attests to its utility. 

The second strategy uses combinations of different antibodies coupled to fluorochromes with distinct emission maxima (5,9). The most relevant fluoro-chromes for combined antigen detection are fluorescein isothiocyanate (FITC abs. max. 494 nm, emiss. max. 517 nm), rhodamine isothiocyanate (TRITC ... 

Extending the utility of fluorescence to various nonfluorophores is achieved via chemical derivatization methods, also termed labeling or tagging methods (Reaction 11.2). Numerous commercial fluorescent tags are available with disparate reactive functional groups. For example, derivatives of fluorescein, fluorescein isothiocyanate (FITC) are reactive toward nucleophiles such as amines and sulfhydryl groups. 

Fluorescein isothiocyanate (FITC)-conjugated annexin V is available from many different suppliers. In the present study, it was produced in house as previously described (30). Protect from light and store at +4°C. 

Abnova FISH probes and Dako FISH probes are labeled with fluorescein isothiocyanate (FITC) and Texas Red (sulforhod-amine lOI acid chloride) haptens, and the probe hybridization sites can be visualized with a BISH detection kit including anti-FITC and anti-Texas Red antibodies. 

The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate- (FITC)-labeled insulin and unlabeled insulins from several species were measured using CE with laser-induced fluorescence detection (CE-LIF) (9). Kd determinations were made by separating free FITC-labeled insulin and its complex with the antibody in equilibrated solutions in 6 s or less (Fig. 3). Dissociation and association rates for insulin, FITC-insulin, and the antibody are fast enough to reach equilibria in less... 

Add 33 pi of fluorescein isothiocyanate (FITC tetramefliylrho-damine isothiocyanate, TRITC, or another fluorescent dye isothiocyanate derivative is used the same way), 50 mg/ml in DMF, to 1 ml of 5 mg/ml IgG in Soln. A. Shake at RT protected from light for 1 h. Remove surplus FITC and its hydrolysis products on a Sephadex G-25 column, equilibrated with PBS. The conjugate appears in the void volume and should be concentrated by ultrafiltration. Add sodium azide to a final concentration of 0.02% (w/v) and glycerol up to 10% (w/v) and store at 4 °C. 

N-hydroxysuccinimide (NHS) (TBAHS, 1 M Na2C03, CH2CI2, r.t., 4h) which afforded glycosyloxysuccinimide derivative 120 in 76% yield (Scheme 21). Interestingly, reacting excess of 120 with tris(2-aminoethyl)amine gave exclusively and in quantitative yield divalent intermediate 121. Further treatment of amine 121 with fluorescein isothiocyanate (FITC) provided heterobifunctional neoglycoconjugate 122 in 62% yield. 

---