FITC-Dextran (MW 4000) (solution)

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

1. Solution preparation
^[2] 1.1 Preparation of working solution
Dilute to 0.1 mg/mL with PBS or serum-free cell culture medium (optimized according to the experiment).
Note: The working solution should be prepared and used immediately, and protected from light.
Storage: The stock solution, store at -20°C or -80°C away from light and avoid repeated freezing and thawing.
Note: Sterilize by filtration through a 0.22 μm filter.

2. Cell staining
Labeling of cells^[1]:
For use with apoptotic HeLa cells and human peripheral blood mononuclear cells (PBMC) (viable HeLa and PBMC can not be stained by FITC-Dextran).
2.1. Incubate cells at 43.5°C for 1 hour and at 37°C for 8 hours to induce apoptosis.
2.2. 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 4000) (the final concentration of PI and FITC-Dextran (MW 4000) is 7.5 μM and 1.13 μM, respectively).
2. 3. Incubate cells for 25 min at room temperature in the dark.
2. 4. Take the labeled cells with 3 mL of medium and centrifuge for 10 min at 500 g.
2. 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 4000) : Ex=495 nm, Em=525 nm).


Paracellular permeability measurement^[4]
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.

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