Millicell ers resistance meter

iBREC (5 × 10³) were seeded on fibronectin-coated membrane inserts (Transwell permeable supports, 0.33 cm² polyester membrane, pore size 0.4 μm, 4 × 10⁶ pores/cm², Corning) and cultivated in ECGM until confluence was reached three to four days later. The culture medium was then replaced by serum-reduced culture medium (SRM; same as ECGM but without hEGF and containing 0.25% FBS and 1 μg/ml fibronectin) for one day before sitagliptin (final concentrations: 10 nM or 1000 nM) was added to both chambers for an additional three days. Transendothelial electrical resistances (TEER) across the cell layers were measured with hand-held chop stick electrodes and a Millicell ERS resistance meter (Millipore, Schwalbach, Germany) at indicated time points [18 (link), 21 (link), 25 (link), 32 (link)]. To avoid temperature-induced changes in the TEER, plates were kept on a warm plate at 37°C during measurements [33 (link)]. Normalized TEER values (n ≥ 4 for each condition) were calculated in relation to those measured in SRM just before addition of effectors.

Mouse and human EDMs were prepared as described in the previous section. EDMs were differentiated for 2 d before treatment with various chemical activators of AMPK, that is, metformin (1 mM) and, A-769662 (100 μM) for 16 h. Cultures were then challenged with either microbial products, that is, LPS (500 ng/ml) and H₂O₂ (100 μM) or live microbes (E. coli K12). On the day of infection, the medium in the basolateral part was also replaced with fresh 5% medium. Trans-epithelial electrical resistance (TEER) was measured using an epithelial voltohmmeter Millicell-ERS resistance meter (Millipore) before and at specific time points after the treatment (0, 4, 8, and 24 h). The supernatant was collected from the basolateral and apical part of the transwell for cytokine analysis, and the cells were collected for RNA extraction followed by expression of target genes by qRT-PCR.