Evom2 voltohmmeter

TEER was performed using an EVOM2 voltohmmeter (World Precision Instrument, Sarasota, FL) connected to an ENDOHM-24SNAP measurement chamber (World Precision Instrument). TEER of RPE monolayers was measured after 1 week and 4 weeks of culture on 3 Transwell membranes.

To assay RPE barrier function, RPE cells were seeded onto 0.33‐cm² Millicell hanging cell culture inserts. At 2 weeks and 6 weeks post‐seeding, transepithelial electrical resistance (TEER) was measured using the EVOM2 voltohmmeter with the STX3 electrode set, following the manufacturer's instructions (World Precision Instruments).

Immortalized human bronchial epithelial cells, 16HBE14o (16HBE), were provided by Dr. Dieter Gruenert (University of California, San Francisco). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, HEPES, and penicillin-streptomycin antibiotics. To study epithelial barriers, cells were grown on collagen-coated Transwell-permeable supports (Corning, Tewksbury, MA) under liquid-liquid conditions. Primary human bronchial epithelial cells isolated from the lungs of pediatric donors were grown on Transwell membrane inserts under air-liquid interface (ALI) conditions as previously described (98 (link)). Transepithelial electric resistance (TEER) was evaluated using an EVOM2 voltohmmeter (World Precision Instruments, Sarasota, FL) and shown as % change from t = 0 as per previous studies (103 (link)). Experiments were performed when the polarized monolayers had a TEER reading >500 Ω x cm², which occurred 7 days after plating for 16HBE and 6 wk for the primary cells (3–4 wk after maintaining primary cells in ALI).

Human intestinal epithelial T84 cells were grown and maintained in DMEM nutrient mixture F-12 ham (DMEM F-12) media (Gibco, Grand Island, NY) as previously described (32 (link)). Cells were plated on permeable transwell inserts (Costar, Cambridge, MA) and grown to confluency and high resistance (>1,000 Ω•cm2). Agonist-stimulated short circuit currents (Isc) were measured in Hank’s balanced salt solution (Sigma-Aldrich) on the apical side using an EVOM2 voltohmmeter (World Precision Instruments, Sarasota, FL). Measurements were taken before fecal solutes were added, 30 minutes post-addition of fecal solutes, and then once an hour for four hours. Cl- secretory responses are expressed as a change in short circuit current (ΔIsc) as previously described (33 (link)).

On day 8 of the iBMEC differentiation, 2e6 cells were plated on 0.4 μm transwells (Corning) in hESCR1 with fresh RA. As a quality control metric to ensure barrier formation, TEER measurements were taken every 24 h following subculture on day 8 for 5 days using an EVOM2 voltohmmeter with STX2 probes (World Precision Instruments). TEER measurements were normalized to resistance (Ω × cm²) across a collagen/FN1-coated transwell with an equal volume of media, containing no cells.

Epithelial barrier formation and integrity were determined by measuring the resistance to an ion current across the epithelium. TEER measurements were taken using the EVOM2 Voltohmmeter with STX2 chopstick electrodes (World Precision Instruments, Florida). Cell inserts were washed with PBS before the addition of pre-warmed HBSS. TEER (Ω cm²) values were taken and calculated by using Eq. 1: