FITC-Dextran (MW 3000-5000)

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).

In Vitro

Labeling of cells:
For use with apoptotic HeLa cells and human peripheral blood mononuclear cells (PBMC) (viable HeLa and PBMC can not be stained by FITC-Dextran).
1. Incubate cells at 43.5°C for 1 hour and at 37°C for 8 hours to induce apoptosis.
2. Suspend the cells in 100 μL of medium, and mix in Q-prep tubes with 10 μL of propidium iodide (PI), 10 μL of FITC-Dextran (MW 3000-5000) (the final concentration of PI and FITC-Dextran (MW 3000-5000) is 7.5 μM and 1.13 μM, respectively).
3. Incubate cells for 25 min at room temperature in the dark.
4. Take the labeled cells with 3 mL of medium and centrifuge for 10 min at 500 g.
5. Take centrifuged cells with 1 mL of medium and use flow cytometry or fluorescence microscopy analyze (PI: Ex=500 nm, Em=600 nm; FITC-Dextran (MW 3000-5000): Ex=495 nm, Em=525 nm).


Paracellular permeability measurement
1. Add FITC-dextran (0.1 mg/mL) to the basal media in the transwell chamber.
2. Collect media from the transwell insert after 15 min.
3. Measure the fluorescence signal (Ex=485 nm, Em=538 nm).
4. Calculate FITC-dextran concentration based on fluorescence intensity.
5. Calculate permeability.

In Vivo

For intestinal barrier function assay
1. Fast mice for 4 h.
2. Orally gavage mice with FITC-Dextran MW 3000-5000 (0.6 mg/g).
3. Measure fluorescence intensity of plasma in 4 h (excitation nm/emission 520 nm).