FITC-PEG400-N3

Application 1: Subcellular Localization of FITC-N₃ (Confocal Laser Scanning Microscopy)^[1]
Cell lines: PC3, DU145, HeLa, DLD, MCF7, WI38, aT3
Reagents: FITC-N₃, MitoTracker (Molecular Probes), Hoechst 33342 (Molecular Probes), 4% paraformaldehyde
Instrument: LSM510 Confocal Laser Scanning Microscope (Carl Zeiss)
Procedure:
(1) Seed cells into 12-well plates containing poly-D-lysine-coated coverslips and culture for 2 days.
(2) Add MitoTracker to a final concentration of 66 nM, and incubate at 37 °C for 30 min to label mitochondria.
(3) Add FITC-N₃ to a final concentration of 10 μmol/L, and incubate at 37 °C for 10 min.
(4) Discard the culture medium, and fix cells with 4% paraformaldehyde for 10 min.
(5) Mount the coverslips, and counterstain cell nuclei with Hoechst 33342 (10 μg/mL).
(6) Observe the subcellular distribution of fluorescent labels under the LSM510 Confocal Laser Scanning Microscope.
Results: FITC-N₃ colocalizes with MitoTracker signals in prostate cancer cells (PC3, DU145) and mainly distributes in mitochondria; no obvious fluorescent signals are observed in non-prostate-derived cells.
Application 2: Labeling Alkyne-Modified Sepharose 4B with FITC-N₃ (CuAAC Click Chemistry)^[2]
Materials: Alkyne-modified Sepharose 4B, FITC-N₃ (prepared by reacting fluorescein isothiocyanate with excess 11-azido-3,6,9-trioxaundecan-1-amine in DMF), CuSO₄, sodium ascorbate
Instruments: QuantaMaster C-60/2000 Fluorescence Spectrophotometer (Photon Technology International)
Procedure:
(1) Take 100 μL of alkyne-modified Sepharose 4B, and mix it with 25 μL of FITC-N₃ solution in a mixture of 460 μL water and 40 μL methanol.
(2) Add 10 μL of 100 mM CuSO₄, 50 μL of 100 mM sodium ascorbate, and 440 μL of water sequentially.
(3) Stir the reaction at room temperature in the dark for 48 h.
(4) After the reaction, wash the gel thoroughly to remove unbound FITC-N₃.
(5) Measure the fluorescence emission of the gel using a fluorescence spectrophotometer (excitation wavelength: 494 nm), and maintain stable gel suspension with the built-in magnetic stirrer during the measurement.
Results: The labeled alkyne-modified Sepharose 4B shows intense characteristic fluorescence emission typical of fluorescein, while the unmodified Sepharose exhibits extremely low background signal, indicating successful terminal alkyne modification and effective conjugation of azide-containing molecules to alkyne-modified Sepharose via CuAAC.

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