5(6)-FITC

Guidelines (The following is our recommended protocol. This protocol only provides guidance and should be modified according to your specific needs).
Preparation of stock solution^[1]
Prepare a 1 mg/mL 5(6)-FITC stock solution in acetone or DMSO and store at 0°C.
Preparation of working solution: Prepare a 2.5 pg/mL solution in phosphate-buffered saline (PBS) and adjust the pH to 7.8.
5(6)-FITC for flow cytometry detection^[1]
(1) Cell preparation: Wash cells from solid nutrient agar plates with 10 mL PBS, centrifuge at 2000 g for 30 min, then resuspend in 10 mL 70% ethanol and incubate at 4°C.
(2) Staining: Collect 3×10⁵ cells by centrifugation, resuspend in 2.5 pg/mL 5(6)-FITC, and stain at 4°C for 45 min.
(3) Detection: Analyze the proportion of green fluorescence using a flow cytometer.
5(6)-FITC for protease assay^[2]
(1) Dissolve 1 g casein in 100 mL solution containing 50 mM calcium carbonate and 150 mM sodium chloride at pH 9.5. The solution may first be heated to boiling and then cooled to room temperature.
(2) Add 40 mg 5(6)-FITC and stir gently at room temperature for 1 h.
(3) Remove free 5(6)-FITC by dialysis, first against 2 L water containing activated charcoal at 4°C, then against 50 mM Tris buffer at pH 8.5, and finally against 50 mM Tris buffer at pH 7.2.
(4) Adjust the protein concentration to 0.5% using 50 mM Tris buffer at pH 7.2. Aliquot the substrate into 5 mL portions and store frozen at -20°C, stable for up to 1 year.
(5) Determine the number of FITC residues per casein molecule (0.65 FITC residues per molecule) by measuring substrate absorbance at 490 nm and protein content using the Lowry method.
(6) Perform reactions using FITC-casein. The reaction mixture contains 10 μL enzyme, 20 μL assay buffer, and 20 μL 0.5% FITC-casein.
(7) Chymotrypsin and trypsin: assay buffer is 100 mM Tris buffer at pH 7.8 containing 10 mM CaCl₂; or 100 mM phosphate buffer at pH 7.8 containing 150 mM NaCl.
(8) Subtilisin: assay buffer is 20 mM phosphate buffer at pH 7.6 containing 150 mM NaCl.
(9) Thermolysin: assay buffer is 100 mM Tris buffer at pH 7.8 containing 10 mM CaCl₂.
(10) Elastase: assay buffer is 20 mM borate buffer at 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. For incubations longer than 3 h, add 0.2% sodium azide to prevent bacterial growth.
(12) Terminate the reaction by adding 120 μL 5% trichloroacetic acid (TCA) and mixing thoroughly. Leave the tubes at room temperature for at least 1 h, then store overnight at 4°C. Precipitate TCA-insoluble proteins by centrifugation for 5 min. Remove 60 μL supernatant and dilute to 400 μL or 3 mL with 500 mM Tris buffer at pH 8.5, mixing thoroughly to ensure the entire sample reaches the appropriate pH.
(13) Fluorescence measurements are performed with excitation wavelengths of 365 nm or 490 nm and an emission wavelength of 525 nm. Use the average of replicate measurements. Calculate the correlation coefficient r and the standard error S of the regression line using single values.
(14) Cleavage of FITC-casein by trypsin, subtilisin, chymotrypsin, elastase, and thermolysin is linearly related to 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 min at 22°C to isolate platelet-rich plasma.
(2) Platelet collection: Centrifuge PRP at 1800 × g for 10 min at 22°C to collect platelets. Contamination by red and white blood cells is less than 0.5%.
(3) Platelet washing: Wash platelets twice with PBS by centrifugation at 1800 × g for 10 min at 22°C, then resuspend in PBS.
(4) Purification of human IgG: Human IgG is purified from normal human serum by DEAE-cellulose chromatography.
(5) Use FITC-labeled goat anti-human IgG antibody: total protein concentration 6.2 mg/mL, fluorescein/protein molar ratio 2.6, and specific antibody concentration 1.6 mg/mL.
(6) Add FITC-labeled goat anti-human IgG antibody to 0.5 mL platelet suspension containing 8.0 × 10⁸ cells/mL to a final concentration of 1.0 × 10^-7 M. After incubation at 22°C for 20 min, wash platelets twice with PBS and resuspend in PBS. Then add SDS (1.5 mg/mL), and determine bound anti-human IgG antibody by fluorescence measurement with excitation at 493 nm and emission at 516 nm.
5(6)-FITC conjugation with PAMAM G4^[4]
(1) Dissolve 30 μL (3 mg) PAMAM-G4 dendrimer in 10 wt% methanol solution and add to 250 μL 0.1 M phosphate buffer (pH 8.5).
(2) Incubate the mixture with 1 mg 5(6)-FITC dissolved in 60 μL dry DMSO at room temperature for 1 h.
(3) Purify FITC-conjugated PAMAM-G4 dendrimers by gel filtration using a PD-10 desalting column containing Sephadex G25. Use 0.1 M phosphate buffer (pH 8.5) as the mobile phase and collect 0.5 mL fractions.
Notes
1. 5(6)-FITC is sensitive to light and moisture. Prepare 5(6)-FITC solutions immediately 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 reduces labeling efficiency.
3. Avoid using buffers containing primary amines (e.g., Tris, glycine) or ammonium ions, as they compete with proteins for labeling reactions.
4. This product is intended for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food, or pharmaceutical use.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.