Assay of Endothelial Monolayer Permeability- FITC-Dextran Method
Preparation of filter membrane inserts
To prepare 12 units of polycarbonate filter inserts,
1. Mix 30 L of fibronectin stock solution and 1 mL of complete growth media
2. Place 80 L of diluted fibronectin solution on each filter insert [Tilt the well plate
several times to make sure that all inserts are covered with fibronectin solution]
* Final fibornectin concentration: 2.1g/cm2 insert
3. Incubate the well plate treated with fibronectin solution at 37 C for at least 30 min,
but not longer than 1 hr.
4. Aspirate extra fibronectin solution very carefully before seeding the cells.
* Polycarbonate filter membrane insert: Transwell, Polycarbonate Membrane, 0.4 m
pore size, 12 mm diameter, 12 units/12-well plate [Costar #3401]
* Preparation of fibronectin solution solution : 1 mg/mL sterile PBS
Seeding the cells on filter membrane inserts
1. Seed 0.5 mL of cells into each chamber consisting of the apical part (upper chamber)
containing the filter membrane insert.
* Cell density : one T-75 culture flask  12 ml of cell suspension in complete
growth media  count the cells using hematocytometer  adjust the cell density to
2.0  105 cells/mL
2. Add 1.5 mL of complete growth media into each well consisting of the basolateral
part (lower chamber).
3. Incubate the cells for 4 days in CO2 incubator.
4. Change the media and incubate the cells for additional 3 days.
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Dr. Lee’s Lab
Treatment of the cells with drugs
1. Aspirate the media gently and wash the inserts twice with basic culture media.
2. Add experimental media with drugs [0.5 mL for upper chamber and 1.5 mL for lower
chamber] and incubate the plate in CO2 incubator for 24 h.
Measurement of permeability
1. Using sterile tweezers, pick up each insert, aspirate the media gently, wash them with
HBSS twice, and transfer inserts to the other fresh 12-well plate.
* Preparation of HEPES buffered salt solution (HBSS), 100 ml : 0.6 g HEPES (25
mM), 0.7 g NaCl (120 mM), 40 mg KCl (5.4 mM), 26.5 mg CaCl2 (1.8 mM), 0.21 g
NaHCO3 (25 mM), 0.27 g Glucose (15 mM)  adjust pH 7.4 with NaOH  filter
sterilization
2. Add 0.5 mL of HBSS containing FITC-Dextran (70 kD, final concentration; 1.0
mg/mL) into upper chamber and 1.5 mL of HBSS into lower chamber.
3. Incubate for 1 hr at 37 C
4. Determine the transferred FITC-Dextran concentration from each lower chamber
using a “Fluorescence Multi-well Plate Reader” with excitation and emission
wavelengths of 485 nm and 530 nm, respectively.
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Dr. Lee’s Lab