5(6)-FITC

5(6)-FITC (Fluorescein 5(6)-isothiocyanate) is an amine-reactive derivative of a fluorescent dye, characterized by high absorbance and excellent fluorescence quantum yield. The isothiocyanate group of FITC can react with various functional groups on proteins, including amines, thiols, imidazoles, tyrosines and carbonyls, enabling the labeling of proteins such as antibodies and lectins. 5(6)-FITC has a wide range of applications, including flow cytometry, immunofluorescence, protease assays and conjugation. The maximum excitation/emission wavelengths are 492/518 nm^[1][2][3][4]. In Vitro:Stock Solution Preparation^[1]
Prepare a 1 mg/mL 5(6)-FITC stock solution in acetone and store at 0°C.
Working Solution Preparation^[1]
Prepare a 2.5 pg/mL solution in phosphate-buffered saline (PBS) with the pH adjusted to 7.8.
Use of 5(6)-FITC for Flow Cytometry^[1]
(1) Cell preparation: Wash cells from solid nutrient agar plates with 10 mL PBS, centrifuge at 2000g for 30 min, then resuspend in 10 mL of 70% ethanol and incubate at 4°C.
(2) Staining: Take 3×10^5 cells and centrifuge them, resuspend the cells in 2.5 pg/mL 5(6)-FITC, and stain at 4°C for 45 min.
(3) Analysis: Analyze the proportion of green fluorescence using a flow cytometer.
Use of 5(6)-FITC for Protease Assay^[2]
(1) Dissolve 1 g casein in 100 mL of a solution containing 50 mM calcium carbonate and 150 mM sodium chloride, with a pH of 9.5. The solution can be heated to boiling and then cooled to room temperature.
(2) Add 40 mg 5(6)-FITC and gently stir at room temperature for 1 h.
(3) Remove free FITC by dialysis, first against 2 L of water containing activated charcoal at 4°C, then against 50 mM Tris buffer, pH 8.5, and finally against 50 mM Tris buffer, pH 7.2.
(4) Adjust the protein concentration to 0.5% using 50 mM Tris buffer, pH 7.2. Aliquot the substrate into 5 mL portions and store frozen at -20°C; it is stable for 1 year.
(5) Determine the number of FITC residues per casein molecule by measuring the absorbance of the substrate at 490 nm and the protein content using the Lowry method, which should yield 0.65 FITC residues per casein molecule.
(6) Perform the reaction using FTC-casein. The reaction mixture contains 10 μL enzyme, 20 μL assay buffer, and 20 μL of 0.5% FTC-casein.
(7) For chymotrypsin and trypsin: the assay buffer is 100 mM Tris buffer, pH 7.8, containing 10 mM CaCl2; or 100 mM phosphate buffer, pH 7.8, containing 150 mM NaCl.
(8) For subtilisin: the assay buffer is 20 mM phosphate buffer, pH 7.6, containing 150 mM NaCl.
(9) For thermostable protease: the assay buffer is 100 mM Tris buffer, pH 7.8, containing 10 mM CaCl2.
(10) For elastase: the assay buffer is 20 mM borate buffer, pH 8.8, containing 150 mM NaCl.
(11) After thorough mixing, incubate the solution in a shaking water bath at 37°C for 5 min to 24 h. If the incubation time exceeds 3 h, add 0.2% sodium azide to prevent bacterial growth.
(12) Terminate the reaction by adding 120 μL of 5% trichloroacetic acid (TCA) and mix thoroughly. Let the tubes stand at room temperature for at least 1 h. Recover the tubes and store overnight at 4°C. Centrifuge for 5 min to precipitate TCA-insoluble proteins. Take 60 μL of the supernatant, dilute it to 400 μL or 3 mL with 500 mM Tris buffer, pH 8.5, and mix well to ensure that the entire sample reaches the appropriate pH.
(13) Measure fluorescence using an excitation wavelength of 365 nm or 490 nm and an emission wavelength of 525 nm. Calculate the average of duplicate measurements. Use individual values to calculate the correlation coefficient r and the standard error S of the regression line.
(14) The cleavage of FTC-casein by trypsin, subtilisin, chymotrypsin, elastase, and thermostable protease shows a linear relationship with the enzyme concentration.
Immunofluorescence Detection of FITC-Labeled Antibodies on Platelets^[3]
(1) Preparation of platelet-rich plasma (PRP): Centrifuge blood at 180 × g for 8 minutes at 22°C to separate PRP.
(2) Platelet collection: Centrifuge PRP at 1800 × g for 10 minutes at 22°C to collect platelets. Contamination with red and white blood cells should be less than 0.5%.
(3) Platelet washing: Wash the platelets twice with PBS at 22°C by centrifuging at 1800 × g for 10 minutes, then resuspend the platelets in PBS.
(4) Purification of human IgG: Human IgG is purified from normal human serum by DEAE-cellulose chromatography.
(5) Use of FITC-labeled goat anti-human IgG: The total protein concentration is 6.2 mg/mL, with a fluorescein/protein molar ratio of 2.6, and the specific antibody concentration is 1.6 mg/mL.
(6) Add FITC-labeled anti-human IgG goat antibody to 0.5 mL of a platelet suspension containing 8.0 × 10⁸ cells/mL to a final concentration of 1.0 × 10^-7 M. Incubate at 22°C for 20 minutes, wash the platelets twice with PBS, and resuspend in PBS. Then add SDS (1.5 mg/mL) and measure the bound anti-human IgG antibody by fluorescence, with an excitation wavelength of 493 nm and an emission wavelength of 516 nm.
Conjugation of 5(6)-FITC with PAMAM G4 Dendrimers^[4]
(1) Dissolve 30 μL (3 mg) of PAMAM-G4 dendrimers in a 10 wt% methanol solution and add to 250 μL of 0.1 M phosphate buffer saline (pH 8.5).
(2) Incubate the mixture with 1 mg of 5(6)-FITC, which is dissolved in 60 μL of dry DMSO, for 1 hour at room temperature.
(3) Purify the 5(6)-FITC-conjugated PAMAM-G4 dendrimers by gel filtration using a PD-10 desalting column containing Sephadex G25. Collect fractions of 0.5 mL using 0.1 M phosphate buffer saline (pH 8.5) as the mobile phase.
Precautions
1. 5(6)-FITC is sensitive to light and humidity. Prepare the 5(6)-FITC solution just before use and discard any unused portion.
2. Low concentrations of sodium azide (≤3 mM or 0.02%) or thimerosal (≤0.02 mM or 0.01%) do not significantly interfere with protein labeling; however, 20-50% glycerol can reduce labeling efficiency.
3. Avoid using buffers containing primary amines (e.g., Tris, glycine) or ammonium ions, as they compete with the proteins to be labeled.
4. This product is for research use only by professional personnel and is not intended for clinical diagnostics or therapeutics, nor for food or drug use.
5. For your safety and health, please wear a lab coat and disposable gloves when handling the product.