Millicell ers 2

To evaluate the monolayer integrity, Caco-2 cells were cultured into 24-well Transwell^® inserts (0.4 μm diameter pores) (Corning, Massachusetts, United States), seeded at a density of 5 × 10⁴ cells/transwell. Transepithelial Electrical Resistance (TER) was measured using an epithelial tissue voltohmmeter (Millicell ERS-2, Merk, Darmstadt, Germany). The experiment started 28 days after the seeding on filters when Caco-2 cells reached a TER value >600 Ω cm² which indicates adequate monolayer integrity and differentiation.

The TEER values of the human iPS cell-derived brain microvascular endothelial cells incubated on the 24-well Transwell inserts were measured using Millicell ERS-2 (Merk-Millipore). To calculate the TEER (Ωxcm² [the surface area of the insert, 0.3 cm²]), the resistance value of an empty filter coated with collagen IV and fibronectin was subtracted from each measurement.

BBB barrier function was evaluated by measuring TEER. The resistance values (Ω) were measured using a Millicell ERS-2 (Merk Millipore). The TEER values were calculated as previously described [10 (link), 11 (link)]. We confirmed the value of TEER was peaked on day 10. Therefore, all experiments were performed on day 10.
The cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma) supplemented with D-glucose and HEPES (DPBS-H). DPBS-H containing 10 μg/mL sodium fluorescein (NaF, Sigma) as a paracellular marker was then added into the upper chamber for 30 min at 37 °C. After incubation, the medium in the lower chamber was collected and then the concentration of NaF in the medium was measured using a fluorescence multi-well plate reader (Genios, TECAN).

TEER values of human iPS-EC monolayers or HUVEC monolayers on Transwell inserts (24-well plates) were measured using Millicell ERS-2 (Merk-Millipore). To calculate TEER (Ω (0 ohrms)×cm² (the surface area of the insert, 0.3 cm²)), the resistance of the fibronectin-coated insert without cells (blank resistance) was always subtracted from the resistance of cell cultures (sample resistance) [2 (link),5 (link)]

Transepithelial electrical resistance of primary CPE cells was performed using Millicell ERS‐2 (MERS00002; Millicell ERS‐2 Voltohmmeter; Millipore). Primary CPE cells were trypsinized, and equal number of cells (10⁶ cells/well) were added to laminin‐coated 12‐well transwell system (CLS3460‐48EA, Sigma). The growth medium was replaced every 48 h. TEER readings were measured every 24 h, according to manufacturer's instructions. In brief, the probe was sterilized by incubating the electrode in 70% ethanol for 15 min and allowed to air dry. Next, the electrode was placed in growth medium for 15 min to equilibrate, followed by TEER measurement of all wells. The blank value (empty wells) was subtracted from the sample readings and multiplied by the effective membrane diameter of the transwell filter. The obtained electrical resistance was expressed in units of Ω.cm².

Stability of the monolayers was assessed using phase contrast microscopy (DMi1, Leica), TEER measurements, and P_app assays. The details of each technique have been described elsewhere [39 (link),40 ]. Briefly, the TEER (Ω*cm²) was measured daily using epithelial Volt-Ohm Meter (Millicell ERS-2, Millipore AG, Burlington, MA, USA), subtracting the blank value measured in Ω and multiplying by the cell culture surface area (0.33 cm²) [37 (link)]. The P_app (cm/s) of 4 kDa FITC-dextran was determined on day 1, 3, and 5 of culture by measuring the fluorescence intensity of the basolateral culture medium over 100 min following apical application of 0.5 mg/mL FITC-dextran to each well. The measurements were obtained with excitation and emission wavelengths of 495 and 535 nm, respectively, using a SpectraMax i3x microplate reader (Molecular Devices) [37 (link)]. The monolayers were considered stable once cells grew to confluence and TEER and P_app values reached a plateau. At least two technical replicates per experiment and more than three biological replicates were assessed to ensure the repeatability. Subsequent experiments were conducted using monolayers which had reached a stable state.