Evom2 epithelial voltmeter

The integrity of monolayer bEnd.3 cells was evaluated by transepithelial electrical resistance (TEER). bEnd.3 cells were plated on the top of Transwell chambers and the TEER was monitored using an EndOhm Chamber connected to an EVOM2 epithelial voltmeter (World Precision Instrument), according to the instructions of the manufacturer. TEER value of the cell layers was obtained through sample TEER subtracting the background TEER values (the resistance of bank filters). TEER monolayer (Ω cm²) = [TEER total (Ω) − TEER blank (Ω)] × 4.67 (cm²).

Cell culture inserts (transwells) with a density of 1 × 10⁵ HLVMEC cells per well were overlaid with 125 μg mL⁻¹ growth factor-reduced Matrigel (diluted in HamF10) and placed in a 24-well plate, at 37 °C and 5% CO₂, and incubated for 24 h. For each sample, the assay was performed in duplicate. After incubation, the medium was replaced with fresh medium containing magnetic nanoparticles labeled with fluorescein (fSi–Fe₃O₄), at a concentration of 1 mg mL⁻¹. To evaluate the integrity of the monolayer as a BBB model the transendothelial electrical resistance (TEER) was measured using an EVOM2 epithelial voltmeter with an STX2 electrode (World Precision Instruments) at 12.5 Hz, as reported previously.^{60 (link)} These measurements were taken during three stages of the test: before addition of the magnetic nanoparticles, and after 24 and 48 h of incubation with them. After exposure to the magnetic nanoparticles, the culture medium in the bottom of the well was recovered and transferred to a tube and pelleted by centrifugation at 1200 rpm for 3 min. The supernatant was eliminated, and 30 μL of the pellet was transferred to a glass slide for analysis by fluorescence microscopy.