Microplate spectrofluorometer

In vitro endothelial permeability was assessed by quantifying the amount of rhodamine B isothiocyanate-dextran (rhodamine-dextran, average MW ~70,000; Sigma-Aldrich) that passed through the endothelial monolayers. Briefly, endothelial cells (2 × 10⁴) were plated on a 24-well Transwell filter (0.4-μm pore size; Corning), and 10% exosome-depleted FBS-1640 medium with exosomes derived from NPC cells with or without EBV at the concentration of 5 μg ml⁻¹ was added into both upper and lower chambers, and then incubated for 48 h to allow the cells to reach 100% confluence. rhodamine-dextran was then added to the upper chamber at 20 mg ml⁻¹, and 1 h later, the amount of rhodamine-dextran in the lower chamber was determined by measuring the fluorescence intensity using a microplate spectrofluorometer (BioTek, Vermont, USA) at a 544 nm excitation and a 590 nm emission wavelength. The investigator was blinded to the group allocation.