5(6)-FITC (Fluorescein isothiocyanate) is an amine-reactive derivative of fluorescein dye with a wide-range of applications in fluorescence microscopy, flow cytometry and immunofluorescence-based assays such as Western blotting and ELISA, as a label for antibodies and other probes.

Physicochemical Properties

Molecular Formula	C21H11NO5S
Molecular Weight	389.3807
Exact Mass	389.035
CAS #	27072-45-3
Related CAS #	63469-13-6 (hydrochloride)
PubChem CID	18730
Appearance	Yellow to orange solid powder
Density	1.5±0.1 g/cm3
Boiling Point	708.6±60.0 °C at 760 mmHg
Melting Point	>360ºC(lit.)
Flash Point	382.4±32.9 °C
Vapour Pressure	0.0±2.3 mmHg at 25°C
Index of Refraction	1.754
LogP	4
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	1
Heavy Atom Count	28
Complexity	668
Defined Atom Stereocenter Count	0
InChi Key	MHMNJMPURVTYEJ-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H11NO5S/c23-12-2-5-16-18(8-12)26-19-9-13(24)3-6-17(19)21(16)15-4-1-11(22-10-28)7-14(15)20(25)27-21/h1-9,23-24H
Chemical Name	3',6'-dihydroxy-6-isothiocyanatospiro[2-benzofuran-3,9'-xanthene]-1-one
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Cell Assay	- Antibody labeling assay: Antibodies were dialyzed against carbonate buffer (pH 9.0) to adjust the pH. 5(6)-FITC was dissolved in anhydrous solvent and added dropwise to the antibody solution at a molar ratio of antibody:FITC = 1:20–1:50. The mixture was incubated at 4°C for 4–6 hours in the dark, then passed through a gel filtration column to separate labeled antibodies from free 5(6)-FITC [1] - Fab' fragment labeling assay: Fab' fragments with a single free thiol group were prepared. 5(6)-FITC was activated to form a thiol-reactive derivative, then incubated with Fab' fragments in phosphate buffer (pH 7.4) at 25°C for 2 hours in the dark. Unconjugated 5(6)-FITC was removed by dialysis to obtain FITC-labeled Fab' while preserving the thiol group [2] - DNA fluorescence detection assay: 5(6)-FITC was conjugated to DNA primers or nucleotides. The labeled DNA samples were subjected to automated sequence analysis, and fluorescence signals emitted by 5(6)-FITC (excitation ~495 nm, emission ~520 nm) were detected by a fluorescence detector to determine DNA sequences [3]
ADME/Pharmacokinetics	Metabolism / Metabolites Cyanide is rapidly alsorbed through oral, inhalation, and dermal routes and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)
Toxicity/Toxicokinetics	Toxicity Summary Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)
References	[1]. Goding JW. Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods. 1976;13(3-4):215-26. [2]. Fluorescein labeling of Fab' while preserving single thiol. Anal Biochem. 1988 Aug 15;173(1):59-63. [3]. Fluorescence detection in automated DNA sequence analysis. Nature. 1986 Jun 12-18;321(6071):674-9.
Additional Infomation	Fluorescein 5-isothiocyanate is the 5-isomer of fluorescein isothiocyanate. Acts as a fluorescent probe capable of being conjugated to tissue and proteins; used as a label in fluorescent antibody staining procedures as well as protein- and amino acid-binding techniques. Fluorescein isothiocyanate is a chemical compound derived from fluorescein and containing cyanide. It is used in flow cytometry. (L552) Fluorescent probe capable of being conjugated to tissue and proteins. It is used as a label in fluorescent antibody staining procedures as well as protein- and amino acid-binding techniques. - 5(6)-FITC (5(6)-Fluorescein isothiocyanate) is a fluorescent labeling reagent with a fluorescein core structure, existing as a mixture of 5-isomer and 6-isomer [1][2][3] - Its primary function is to conjugate with biomolecules (antibodies, Fab' fragments, DNA, etc.) via isothiocyanate group (-NCS) reacting with amino or thiol groups of target molecules [1][2][3] - It exhibits strong fluorescence with excitation wavelength ~495 nm and emission wavelength ~520 nm, enabling sensitive detection of labeled biomolecules [3]

Solubility Data

Solubility (In Vitro)

DMSO : ≥ 156 mg/mL (~400.64 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5682 mL	12.8409 mL	25.6819 mL
	5 mM	0.5136 mL	2.5682 mL	5.1364 mL
	10 mM	0.2568 mL	1.2841 mL	2.5682 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.