FITC-Heparin

Protocol Guide (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 FITC-Heparin can be used without further purification.

2. Working Solution Preparation
2.1 Diluent: high-glucose medium containing 10% FBS; water.
2.2 Working concentration: 100 µg/mL FITC-Heparin for purified EVs^[1]; 1 µg/mL FITC-Heparin for cell uptake experiments^[2]; 5 µg/mL FITC-Heparin for HGF N-domain binding experiments^[8]; for FITC-Heparin nanoSTAT formulations, the heparin concentration is fixed at 20 U/mL (0.1 mg/mL)^[3].
2.3 Note: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For purified EVs^[1]:
3.1.1 Incubate 10 µg EVs with FITC-Heparin working solution at 4°C overnight.
3.1.2 For co-localization experiments, add red CMTPX-labeled U87-MG-derived EVs to FITC-Heparin and incubate at 4°C overnight.
3.1.3 After incubation, pellet EVs by ultracentrifugation at 100,000×g for 2 h, and resuspend each pellet in 150 µL 1× PBS.
3.1.4 Take 10 µL of each sample and analyze by confocal imaging in duplicate wells.

3.2 For U937 promonocytes^[2]:
3.2.1 Use cultures stimulated to divide after growth arrest with high-glucose medium containing 10% FBS.
3.2.2 Incubate cells with FITC-Heparin working solution for 1 hr and 24 hrs.
3.2.3 Transfer cells to slides by cytocentrifugation.
3.2.4 Air-dry the cytocentrifuged cells and gently fix with Tissue and Cell Fixative (4% Paraformaldehyde, PFA) (HY-DY3003) at room temperature for 10 min.
3.2.5 Apply mounting medium containing DAPI (HY-D2868), then cover with a coverslip.

3.3 For nanoSTAT formulations^[3]:
3.3.1 Prepare nanoSTATs using fluorescently labeled (FITC) heparin.
3.3.2 Prepare analytical samples of intact nanoSTATs, nanoSTATs incubated with thrombin, and free heparin.
3.3.3 Incubate one aliquot of nanoSTATs sample with 500 nM thrombin at 37°C for 30 min. Incubate a parallel nanoSTAT sample without thrombin as a control.
3.3.4 Before detection, load samples onto a size exclusion column pre-equilibrated with PBS.

3.4 For core-shell fiber mats^[4]:
3.4.1 Use core-shell fiber mats composed of cationized Gelatins (HY-Y1365)-coated PCL prepared by coaxial electrospinning.
3.4.2 Investigate the adsorption behavior of FITC-Heparin on these fiber mats.

3.5 For balloon-injured canine iliac artery segments^[7]:
3.5.1 After local or intravenous administration of FITC-Heparin, excise the iliac artery at 2 h or 4 h after administration.
3.5.2 Embed the excised vessel rings in tissue embedding medium, snap-freeze in liquid nitrogen, cut into 13 µm-thick frozen sections, and mount the sections onto slides.
3.5.3 Apply FITC-Heparin locally at 30 units/kg to the injured iliac artery segments. After balloon injury of the common iliac artery, administer FITC-Heparin intravenously at 100 U/kg.

3.6 For isolated HGF N-domain in solution^[8]:
3.6.1 Prepare protein samples in 50 mM sodium phosphate and 100 mM NaCl at pH 6.8.
3.6.2 Use FITC-Heparin working solution in assay buffer containing 100 mM NaCl, 50 mM sodium phosphate, pH 6.8.
3.6.3 Maintain samples in a thermostatted sample holder at 25°C.

4. Controls
4.1 Negative control: incubate FITC-Heparin with 1× PBS without EVs^[1].
4.2 nanoSTAT controls: intact nanoSTATs (without thrombin), thrombin-treated nanoSTATs, free heparin^[3].
4.3 Competition control: add unlabeled heparin to the preformed FITC-Heparin-N-domain complex for displacement; a significant excess of unlabeled heparin is required for 50% displacement, and re-equilibration is rapid; the N-domain has approximately 7-fold higher affinity for FITC-Heparin than for unlabeled heparin^[8].

5. Detection and Analysis
5.1 Instrument types: confocal laser scanning microscope; fluorescence microscope equipped with epifluorescence illumination; laser confocal microscope system equipped with an inverted microscope; fluorescence microscope; analytical FPLC equipped with a UV flow detector; fluorescence microplate reader; fluorescence spectrophotometer.
5.2 Wavelength settings:
5.2.1 FITC-Heparin^[2][7]: excitation with a 488 nm argon ion laser, detection of green emission with BP 505-530 nm; Ex/Em: 492/514 nm
5.2.2 Red-labeled EVs^[5]: excitation at 543 nm using a HeNe laser; detection of red fluorescence emission through a 560-615 filter.
5.2.3 Monitor column eluate absorbance at 488 nm^[3].
5.2.4 Fluorescence measurement: Ex/Em: 485/538 nm^[3].
5.2.5 HGF binding titration: excitation at 460 nm, emission scan from 480-700 nm; excitation spectra monitored at 550 nm emission, scanned from 340-540 nm^[8].

5.3 Result analysis:
5.3.1 FITC-Heparin green fluorescence co-localizes with red-labeled EVs, appearing yellow in merged images; aggregates of varying sizes are observed^[5].
5.3.2 In FPLC, intact nanoSTATs show a sharp peak at 7 mL; after incubation with thrombin, this peak disappears and is replaced by a broad peak at approximately 11 to 17 mL corresponding to free heparin; free heparin is below the detection limit of intact nanoSTATs and is released after thrombin incubation^[3].
5.3.3 For fluorescence measurement, plasma fluorescence is measured over time after administration of FITC-Heparin formulations^[3].
5.3.4 Weak fluorescence of FITC-Heparin is detected at the injured artery 2 h after infusion. Weak fluorescence of FITC-Heparin is detected in smooth muscle cells and subintimal tissue of the injured artery 2 h after systemic and local administration. Adhesion and accumulation of FITC-labeled heparin in the inner layer of the vessel wall are observed 2 h after administration. No fluorescence is observed 4 h after administration of FITC-labeled heparin. Intravenously administered FITC-labeled heparin is retained until 2 h after administration and is almost completely washed out 3 h after administration^[8].
5.3.5 Addition of a saturating amount of N-domain to FITC-Heparin results in a 44% decrease in fluorescein emission intensity.