Ecis system

To evaluate the real-time alteration of the monolayer cell resistance, the ECIS system (Applied BioPhysics, Troy, MI, USA) was applied to compare the impedance values in hBMECs with or without lncRSPH9-4 overexpression seeded on the collagen-coated, gold-plate electrodes in 96-well chanmer slides (96W1E+), as previously described [50 (link)]. Two ECIS parameters, R (Ω), representing the electrical cell–cell contacts, and Rb (Ωcm²), representing the paracellular barrier, were extracted from the continuously recorded impedance spectra to reflect the real-time changes of the monolayer barrier function.

Cell motility was determined using the electric cell-substrate impedance sensing (ECIS) system (Applied Biophysics, Troy, NY) as described previously (Keese et al. 2004 (link)). Cells were grown on ECIS electrode arrays (8W1E). The impedance fluctuations of cell attachment and spread were continuously monitored. An alternating current of 1 μA at 4 kHz was applied between a small sensing electrode (250-μm diameter) and a relatively large counter electrode. At confluence, CSE (20 μg/mL) was added to the culture medium. Experiments were conducted up to 2 days after CSE introduction. Wounding of cells was achieved using a 6V signal at 45 kHz for a duration of 30 sec. Application of this field results in a rapid drop in the impedance of cell layers due to the death of the cells on the electrode. Impedance increases as cells migrate from the perimeter of the electrode inward to replace the wounded cells.

We used Electric Cell-substrate Impedance Sensing (ECIS) method to perform cell proliferation assays. An ECIS system was obtained from Applied BioPhysics, Inc. The ECIS culture-ware used was 96W10idf. Briefly, the protocol was as follows: The cell suspension was prepared first, and then counted. The cell suspension was diluted to 4 × 10³ cells in 200 μl, and added to a 96-well electrode plate. Then, the signal from the cells in the electrode plate was conventionally monitored for 48–72 h. The data were analyzed by the specific software matched to the ECIS system. The experiments were performed in triplicate.

A549 cells were seeded on 8W10E+ ECIS arrays (Applied Biophysics, Troy, NY) at a density of 0.5 x 10⁶ cells/mL. Cells were grown to a confluence as measured by a reading of 10 μF on the ECIS System (Applied Biophysics, Troy, NY). After achieving confluence, A549 cells were subjected to serum starvation and were treated with IL-1β (10 ng/mL) in the presence or absence of AT-RvD1 (100 nM) for 6 hours. Cells receiving resolvins were pretreated with AT-RvD1 for 30 minutes prior to addition of IL-1β or vehicle. Resistance measurements were taken for a period of 48 hours. Three samples were used and 4 measurements were taken at each time point to mitigate inter- and intra- sample variability. Culture medium served as the electrode and barrier function was analyzed as a function of impedance of a cell-covered electrode. A 1-V, 4,000-Hz alternating current signal was supplied through a 1-MΩ resistor to approximate a constant-current source. ECMS 1.0 software (CET, Coralville, IA) was used to measure the in-phase voltage resistance and capacitance. Epithelial barrier function of A549 cells was expressed as TER, which was normalized to a time-zero baseline. Epithelial monolayers that did not achieve a baseline TER of 5,000 Ω or higher were excluded from this study.