Millicell ers electrical resistance system

Cell monolayers in 0.33 cm² transwell inserts were exposed to H₂O₂ in the presence or absence of different forms of Alb. TER was measured using a Millicell-ERS Electrical Resistance System (Millipore, Bedford, MA) as we have previously reported [38 (link)].

The barrier function was evaluated by measuring transepithelial electrical resistance (TER) and unidirectional flux of FITC-inulin.
Measurement of TER: TER was measured as described previously [32 (link)] using a Millicell-ERS Electrical Resistance System (Millipore, Bedford, MA, USA). TER was calculated as Ω·cm² by multiplying it with the surface area of the monolayer. The TER of the polycarbonate membrane in Transwells (approximately 30 Ω·cm²) was subtracted from all readings.
The unidirectional flux of inulin: Cell monolayers in transwells were incubated in the presence of FITC-inulin (0.5 mg/mL) in the basal well. At varying times during the treatments, 50 μL of the apical medium was withdrawn, and fluorescence was measured using a fluorescence plate reader (BioTEK Instruments, Winooski, VT, USA). The flux into the apical well was calculated as the percent of total fluorescence administered into the basal well per hour per cm² surface area.

TER values of epithelial cell monolayers were measured to evaluate the tight junction permeability.^{61 (link)} Briefly, 16HBE14o- cells were seeded into plate inserts (12 mm diameter, 3.0 μm pore size, BIOFIL JET), and the TER of 16HBE14o- monolayers was measured in the presence or absence of SARS-CoV-2 E protein or S protein, using the Millicell-ERS Electrical Resistance System (Millipore, USA). The TER values were obtained by subtracting the intrinsic resistance of the membrane, and multiplied by the surface area of the filter (1.12 cm²). After stimulation, the E protein significantly decreased the TER at a concentration of 50 μg/mL (Supplementary Fig. 5f).

M-like cells were obtained as previously described [45 (link)–47 (link)] with modifications. Briefly, Caco-2 cells (10⁵ cells/filter) were seeded on the upper chamber of a Millicell filter (3.0-μm pore diameter, Millipore, USA) and kept in DMEM as described above for 10 days at 37°C in an atmosphere of 5% CO₂. The lower chamber was also filled with DMEM. During this incubation period, transmembrane electric resistance (TEER) was measured every two days using the Millicell^® ERS (Electrical Resistance System, Millipore), until it reached ~420 mΩ. Afterwards, Raji-B cells (10⁶ cells/mL) were seeded at the Millicell lower chamber and cultured in RPMI-1640, as described above, for 6 days. In parallel, in some filters, Caco-2 cells were kept in monoculture for an additional 6 days (non-differentiated cells). Since galectin-9 is expressed on M cells but not on Caco-2 cell surface [47 (link)], NIH 3T3 (positive control), Caco-2 (negative control) and M-like cells were fixed and incubated with galectin-9 (M-20):sc-19294 (Santa Cruz Biotechnology, INC), followed by Alexa Fluor 488 (donkey anti-goat IgG, Invitrogen) incubation. Green fluorescent cells indicated the presence of galectin-9. Alternatively, donkey anti-goat IgG-TR (Santa Cruz Biotechnology, INC), combined with phalloidin-FITC (Sigma, USA) and DAPI (Molecular Probes, USA) were used.