Preparation of FITC-Dextran Solution
Resuspend FITC-Dextran in phenol-red free MEM warmed to 37 oC to a concentration of 1 mg/mL in a sterile environment. Protect solution from light.
1. Prepare triplicate 12-well collagen coated inserts for monolayer-free positive control within multi-well plate set-up.
2. Expose test samples with compound of interest.
3. Transfer and blot TranswellÒ inserts containing exposed cells to new multi-well plate and add 800 μL phenol red-free MEM to basolateral compartment.
4. Aspirate apical compartment media and replace with 250 μL FITC-Dextran suspension.
5. Protect from light at room temperature for 20 minutes.
6. End FITC-Dextran reaction by transferring TranswellÒ inserts with FITC-Dextran suspension to new multi-well plate.
7. Collect FITC-Dextran suspension basolateral media in 1.5 mL Eppendorf tubes and vortex.
8. Transfer 100 μL aliquots from each Eppendorf tube containing FITC-Dextran basolateral media to Corning™ 96-Well Clear Bottom Black Polystyrene Microplates in triplicate.
9. Transfer 100 μL of phenol-red free MEM to an additional three wells to serve as the blank control.
10. Measure fluorescence intensity on the Polarstar Plate Reader using 490/520 nm excitation/emission maxima.
a. Note: Before measuring fluorescence, use the Polarstar’s “Adjust Gain” setting to adjust all your samples to the well with the highest fluorescence.
11. Export data to Excel for analysis.