FITC-Dextran (MW 4000)

Protocol for BBB permeability in vitro using FITC-Dextran^[1]:

The in vitro study of BBB permeability was performed by using a transwell assay.

1. Fifty thousand bEnd.3 cells were seeded in an insert of the transwell system (0.4 μm pore), which was placed in a 24-well-plate.
2. After endothelial monolayer formed in the insert, the culture media in both insert and lower chamber were replaced by conditioned medium and control medium respectively followed by overnight incubation.
3. FITC-Dextran (0.1 mg/mL) was added to the upper chamber. The fluorescence penetration was detected after incubation for 20 min by a plate reader.
4. The vascular integrity was evaluated as the absorbance differences between insert and lower chamber.

Protocol for cell marking using FITC-dextran ^[2]:

Suitable for apoptotic HeLa cells and human peripheral blood mononuclear cells (PBMC)

1. Apoptosis was induced by incubation at 43.5℃ for 1 h and 37℃ for 8 h.

2. The cells were suspended in 100 μL medium in Q-prep tube mixed with 10 μL propidium iodide (PI),10 μL FITC-Dextran (MW 4000), PI and FITC-Dextran (MW 4000) final concentrations were 7.5 μM and 1.13 μM, respectively.

3. The cells were cultured at room temperature in the dark for 25 minutes.

4. Take 3 mL of labeled cells and centrifuge 500 g for 10 min.

5.The centrifuged cells were added to 1 mL medium and analyzed by flow cytometry or fluorescence microscopy.

(PI: Ex=500 nm, Em=600 nm; FITC-Dextran (MW 4000): Ex=495 nm, Em=525 nm)

Protocol for In vivo intestinal permeability test ^[3]:

1.    For the studies investigating intestinal permeability in obese and lean mice. Mice were fed the assigned diet for 10-16 weeks. mice were weight stratified to the two subgroups, LBM dose or BW dose (lean body weight groups and obese body weight groups). At study end, the mice were 18-24 weeks.

2.    On the study day, Mice fasted for four hours before FITC-Dextran (MW 4000)(dissolved in PBS at a concentration of 125 mg/ml) were administered orally (600 mg/kg) in a randomized order.

3.    Two hours after dosing, mice were anesthetized using isoflurane (induction 5%, maintenance 2% isoflurane, 0.7 L/min N₂O, 0.3 L/min O₂), and retro-orbital blood sampled in K₃-EDTA-coated tubes followed by cervical dislocation.

4.    The samples were centrifuged (4℃, 7 min, 8000 g), and plasma was collected in clear Eppendorf tubes.

Plasma from PBS dosed mice were used for the standard curve. FITC-Dextran concentrations in plasma were analyzed in duplicates using a spectrophotometer.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. Wu S, Wang J, et,al. Programmed cell death 10 increased blood-brain barrier permeability through HMGB1/TLR4 mediated downregulation of endothelial ZO-1 in glioblastoma. Cell Signal. 2023 Jul;107:110683. doi: 10.1016/j.cellsig.2023.110683. Epub 2023 Apr 17. PMID: 37075875.

[2]. Moumaris M, et al. Fluorescein isothiocyanate-dextran can track apoptosis and necrosis induced by heat shock of peripheral blood mononuclear cells and HeLa cells[J]. Open Biological Sciences Journal, 2015, 1(1).

[3]. Voetmann LM, Rolin B, et al. The intestinal permeability marker FITC-dextran 4kDa should be dosed according to lean body mass in obese mice. Nutr Diabetes. 2023 Jan 5;13(1):1. doi: 10.1038/s41387-022-00230-2. PMID: 36604407; PMCID: PMC9816099.