Ecis zθ

Endothelial barrier function was assessed by measuring trans-endothelial electrical resistance (TEER) using the electric cell-substrate impedance sensing system (ECIS Zθ, Applied Biophysics). ECIS plates (96W20idf PET, Applied Biophysics) were pretreated with L-Cystein and coated with 1% gelatin. After taking baseline measurements, endothelial cells with and without knockdown or overexpression were added to the plate in the absence or presence of 10 μM Y-27632 ROCK inhibitor (StemCell technologies, 72302). Multiple frequency/time (MFT) mode was used for real-time assessment of the barrier. Results are expressed as relative resistance at frequency of 4,000 Hz corrected for baseline resistance. ECIS software was used for further mathematical modeling. Using impedance data, this model enables to calculate the cell morphological parameters cell-cell (Rb) and cell-matrix (α) contacts (27 (link), 28 (link)).
In addition, stable endothelial barriers were modulated by addition of 10 μM Y-27632 ROCK inhibitor or 1 μg/mL recombinant SEMA3A (R&D Systems, 1250-S3). Results are expressed as percentage of the average barrier of the endothelial cells measured over 4–5 h before addition of stimuli.

EC monolayer function was measured using ECIS, an electric cell-substrate impedance sensing system (ECIS Zθ, Applied Biophysics, Troy, NY), as previously described (Santaterra et al., 2020 (link)). The system then applies weak alternating currents through the electrode array and continuously measures the ability of the cell monolayer to impede the movement of electrons between adjacent EC (resistance). Briefly, cells were seeded at 2.5 × 10⁵ cells/well on fibronectin-coated (10 µg/mL) eight-well arrays (8WE10, Applied Biophysics) containing interdigitated gold electrodes. ECs were seeded 48 hr before experiments and the resistance started to be recorded after 48 hr. Only wells with resistance >1500 ohms and stable impedance/resistance readings were used. Before stimulation, resistance was continuously monitored for 2 hr to confirm monolayer stability represented by a plateau in the resistance curve. Stimuli (20% v/v pooled plasma in complete medium) was then added to wells under continuous impedance/resistance monitoring for 12 hr. A baseline resistance value was recorded immediately prior to the addition of each stimuli, and results are expressed as a ratio from baseline resistance (normalized resistance).

The electric cell substrate impedance-sensing technique (ECISZΘ, Applied BioPhysics Inc, Troy, NY, USA) was used to determine transendothelial electrical resistance (TER) as a measure of HAoEC barrier integrity. HAoEC were seeded into equilibrated ECIS arrays and grown to confluency for 5–7 days. TER was measured at 4,000 Hz for 1 h to allow stabilization of HAoECs in electrode arrays. Afterwards, cells were treated with 50 ng/ml IFN-γ, 10 ng/ml TNF-α, or 50 ng/ml IFN-γ + 10 ng/ml TNF-α. Changes in endothelial barrier integrity were observed by ongoing measurement of TER at 4000 Hz. Results were analyzed using ECIS, Excel and PRISM software. Experiments were carried out with n = 5.

Electric Cell-substrate Impedance Sensing (ECIS Zθ, Applied BioPhysics, Inc., New York, NY, USA) was used to perform the kinetic analysis of in vitro killing mediated by CAR T cells [24 (link),25 (link)]. JIMT-1 target cells were grown in 8W10E PET 8-well arrays with gold electrodes at the bottom. The complex impedance spectrum of cells adhered to the electrodes was assessed from 1 Hz to 100,000 Hz. The effector/target cell ratio was set at 1:1. Treatment started after 25 h of incubating the cells on the plate when the impedance of the target cells reached a plateau representing a completely covered cell culture surface which is essential for appropriate comparison of various treatments. CAR T cells were compared co-temporally in technical replicates, and two independent experiments were run. Impedance was monitored for 25h. Averaged traces were normalized to impedance measured at the start of treatment, and then normalized impedances at every time point were normalized to the corresponding value in the NT T cell control.

A 96-well electrode array comprised of 20 interdigitated electrode fingers with a total area of 3.92 mm² (Applied Biophysics, Troy, NY) was coated with 20 μg/ml fibronectin, and 4 × 10⁴ to 5 × 10⁴ HUVEC were seeded into each well. Immediately after seeding, the array was placed on an ECIS Zθ (Applied Biophysics) and maintained at 37°C and 5% CO₂. The impedance in each well was measured every 15 min at multiple alternating current (AC) frequencies. HUVEC were provided fresh media at 24 and 48 h after seeding. At 72 h after initial seeding, 0.5 ng/ml IL-1β, syringe-lysed T. gondii tachyzoites at an MOI of 1 or 2, or fresh media were added to the test wells, and the assay was conducted for an additional 24 h. Impedance measured at 4,000 Hz and 64,000 Hz was used to calculate resistance and capacitance, respectively. Resistance was then multiplied by the surface area of the electrical cell-substrate impedance sensing (ECIS) electrodes to calculate transendothelial electrical resistance (TEER) values (61 (link)).