Fitc dextran 40

Confluent HUVECs were treated with or without HGHF for 48 h on type I collagen-coated culture inserts with 0.4-µm pores (BD Bioscience, Franklin Lakes, NJ, USA). After that, 15 µL of 5 mg/ml FITC-dextran 40 (Sigma-Aldrich) was added to the upper chamber and incubated at 37°C for 15, 30, and 60 min. The fluorescence intensity of FITC-dextran diffused into the lower chamber was measured at Ex/Em = 485/590 nm.
Endothelial cell permeability was detected by using an In Vitro Vascular Permeability Assay kit following the manufacturer’s protocol (Millipore, Burlington, MA, USA). After forming a tighter monolayer and exposure to HGHF, the cells were stained with FITC-dextran 40 for 20 min. The amount of FITC-dextran 40 (Aman et al., 2012 (link)) diffused across the endothelial monolayer was observed under a fluorescent microscope with a FITC filter and quantified on a fluorescence plate reader.
Leaked ratio = Transwell lower chamber fluorescence intensity (Ex₄₈₀/Em₅₉₀)/upper chamber fluorescence intensity (Ex₄₈₀/Em₅₉₀).

To determine the difference in the phagocytic function of BV2 and Raw264.7 cells after OGD, these two cell types were incubated with FITC-dextran 40 (TdB Consultancy AB, cat: FD40, Sweden) for 30 min at 4°C or 37°C after OGD. Following removal of FITC-dextran 40 and washing with cold PBS, the cells were coverslipped with Fluoroshield with DAPI (Sigma, F6057-20 ml, USA) and visualized with a fluorescence microscope (Zeiss, Axio Observer Z1, Germany).
For flow cytometry, the cells were trypsinized after OGD and washed twice with PBS. Gating and data analysis was performed using FlowJo software (TreeStar, USA).

In vitro permeability assay was carried out according to the method of Chen et al. 33 (link). HUVECs were grown to confluence on type I collagen-coated culture inserts with 0.4-μm pores (BD bioscience), treated with or without test samples for 18 h. After the incubation, 15 μL of 5 mg/mL FITC-dextran 40 (Sigma-Aldrich) was added to the upper chamber and incubated at 37 °C for 30 min. Fluorescence intensity of FITC-dextran diffused into the lower chamber was measured with the excitation at 485 nm and the emission at 590 nm. Miles vascular permeability assay was carried out using mice according to the method of Zhang et al. 34 . Evans blue dye (200 μL of 0.5% solution in 0.9% NaCl) was injected intravenously into mice. Ten min later, a test sample or PBS as control was injected intradermally into the right and left sides, respectively, of the back skin (50 μL each) or the ears (10 μL each) in each mouse. At 30 min, the animals were euthanized, and the skin area including the entire injection site was removed by punch biopsies. Evans blue dye was extracted from the skin tissues with formamide and measured for absorbance at 595 nm.