Millicell ers system

To test intestinal epithelial barrier permeability, Caco-2 cells were seeded into upper transwell chambers. Using the Millicell-ERS system (Millipore, Burlington, MA, USA) to measure the Transepithelial electrical resistance (TEER).

Transepithelial electrical resistance (TER) of filter-grown Caco-2 monolayers was measured using a Millicell-ERS system (Millipore, Bedford, USA). Cells were seeded on transwell inserts (Corning, Cambridge, USA) with polyethylene terephthalate membrane (0.33 cm², 0.4 μm pore size) at a density of 1.0 ×10⁶ cells/well and monitored daily for about 21 days. When cells reached confluence and completely differentiated, different concentration FA (25, 50, or 100 μM) were added to the apical of the filter for 2 h and then treated with or without LPS. Changes in TER under experimental conditions were expressed as a percentage of the corresponding basal values. Determinations were repeatedly operated on three different sites transwell insert, respectively.
The permeability of the epithelial across Caco-2 cell monolayers was quantified by measuring the flux of the FITC-labeled dextran of molecular mass 4 kDa (FD4; Sigma-Aldrich, St. Louis, USA). In brief, 1 mg/ml of FD4 was added to the apical chamber and the medium was collected from the basolateral chamber after 2 h’s incubation. Fluorescence intensity of FITC was quantified using a fluorescence plate reader (BioTek, Winooski, USA) with excitation at 492 nm and emission at 520 nm.

HMVEC-d (2 × 10⁵ cells/well) in EGM^TM 2MV complete medium was seeded onto transwell inserts (3.0 µM, 6.5 mm insert; Corning Inc., Canton, NY, USA) and confluence was monitored by measuring transendothelial electrical resistance (TEER) across cell monolayers using Millicell-ERS system (Millipore, Billerica, MA, USA) voltmeter with chamber Endohm 6 (World Precision, Saratoga, FL, USA). The resistance values (TEER) were calculated as Ω/cm² (Ohms) by subtracting the resistance value in each point time by the blank membrane resistance (without cells) and considering that resistance is inversely proportional to the area of the membrane (0.33 cm²). HMVEC-d grown in EGM^TM 2MV medium only on transwell inserts were used as time 0. On cell monolayers, 20% heat inactivated serum was added from patients in EGM^TM 2MV and cell permeability was monitored before (time 0) and after 5, 15, 30 min, 1, 2, 3, and 4 h. Data was normalized in relation to cells culture in EGM^TM 2MV only and relative TEER values at each kinetic point for each individual patient were calculated by understanding how many times the TEER value changed over time zero. In addition, we decided to set the threshold for the decrease in TEER (dashed a gray area 0.4 relative to TEER) constituted a “critical zone”, as all our healthy donors had relative TEER values above 0.4 considering at any time between 5 min to 4 h.

Caco-2 cells, seeded on top of transwell chambers, were used to estimate transepithelial cell resistance of the intestinal epithelial barrier using the Millicell-ERS system (Millipore, USA). Two transwells with only culture medium were set as blank control, and the whole measurement process was carried out at a constant temperature. Each transwell was estimated in three different directions, thrice and the average was used to find the transepithelial electrical resistance (TEER), expressed in Ω·cm2. TEER = (measured TEER-blank pore TEER) ×effective membrane area of cell culture compartment

The activity of compound 2b on intestinal cells monolayer permeability was assessed by measuring the transepithelial electrical resistance (TEER) value. Caco-2 cells (0.5 × 10⁵ cells/well) were grown in 12 mm i.d. Transwell inserts (polycarbonate membrane, 0.4 μm pore size) and culture medium was dispensed in the apical (0.5 mL) and basolateral (1.5 mL) compartments of each well. Resistance was measured using a Millicell–ERS ohmmeter (Millicell-ERS system, Millipore, Bedford, MA, USA) as previously reported by Serreli et al., [27 (link)]. Once the cell monolayers were formed, only inserts with TEER values >300 Ω/cm² were chosen for the experiment. Then, compound 2b (final concentration 20 µM) and, to induce an increase in permeability, an oxysterol mixture (final concentration 60 µM) prepared as described by Serra et al. [28 (link)] were added to the culture medium. TEER values were subsequently measured at intervals of 0.25, 0.5, 0.75, 1, 2, 3, 6, 18, 24, and 48 h and are reported as a percentage of the corresponding TEER value at time zero (T = 0).