Millicell ers electric resistance system

HCT-8 were seeded on Milicell culture inserts (PIHP01250, Millipore, Billerica, MA, USA) of 12 mm diameter and 0.4 um pore size (filter area: 1.13 cm) placed on a 24-well plate and grown for about 7–10 days as previously described until a continuous monolayer was achieved. The development of monolayers was monitored measuring the transepithelial electrical resistance (TEER) with a Millicell-ERS electric resistance system (Millipore, Billerica, MA, USA) until TEER values were stable for 2 consecutive days and higher than 1,200 Ω.cm2, which is consistent with cell polarization (Hurley et al., 1999 (link)).

HCT-8 were seeded on Milicell culture inserts (PIHP01250, Millipore, Billerica, MA, USA) of 12 mm diameter and 0.4 um pore size (filter area: 1.13 cm) placed on a 24-well plate and grown for about 7–10 days as previously described until a continuous monolayer was achieved. The development of monolayers was monitored with the electrical resistance (TEER) measured with a Millicell-ERS electric resistance system (Millipore, Billerica, MA, USA) until TEER values were stable for 2 consecutive days and higher than 1,200 Ω.cm², which is consistent with cell polarization (Hurley et al., 1999 (link)).

HGEC and HK-2 co-cultures were done using Millicell cell culture inserts (PIHP01250, Millipore, Billerica, MA, USA). HGEC (5.10⁴) were seeded on the lower side of the Millicell filter (0.4 µm membrane pore size) and allowed to attach during 12–16 h. Then, inserts were inverted, and HK-2 (7.10⁴) was seeded into the upper side. Co-cultures were maintained under growth conditions in HGEC complete medium. To obtain HK-2 and HGEC monocultures, the same method was performed but partner cells were not seeded. The integrity of HGEC and HK-2 monolayers and HGEC/HK-2 bilayers was verified by using a Millicell-ERS electric resistance system (Millipore, Billerica, MA, USA) calibrated for each measurement, as previously described [36 (link)]. To analyze the effects of Stx2 and SubAB on monocultures and cocultures, Stx2 or SubAB or Stx2 + SubAB were incorporated into the bottom compartment (HGEC compartment).