Electric cell-substrate impedance sensing (ECIS) measurements were performed using 8W1E+ electrode arrays on an ECIS Zθ instrument (Applied Biophysics, Troy, NY). The measurements were performed as described previously [25, 26] . A baseline was established using culture medium (400 μL•well -1 ). The resistance was recorded in units of Ω at a frequency of 500 Hz. At 48 hours after transfection, the cells were sub-cultured on an ECIS array. Exposure to hyperoxia or normoxia began when the electrode was covered with a monolayer of cells. The ECIS allows for a sensitive determination of the amount of current passing between cells and the resistance of the barrier (Rb) (in units of Ω cm 2 ). Rb is a robust reporter of barrier function [27] .
Ecis zθ instrument
Manufactured by Applied Biophysics
Sourced in United States
The ECIS Zθ instrument is a device used for measurement and analysis of cellular behavior and adhesion. It utilizes electrical impedance spectroscopy to monitor changes in cell attachment, morphology, and proliferation in real-time. The instrument provides quantitative data on various cellular parameters without the need for labels or dyes.
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Lab products found in correlation
8w10e arrays, by Ibidi (2 mentions)
96w10idf, by Applied Biophysics (1 mentions)
Purecol type 1 bovine collagen solution, by Advanced BioMatrix (1 mentions)
Ecis software, by Applied Biophysics (1 mentions)
Embryomax, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Gfr matrigel matrix, by BD (1 mentions)
Actinred 555 readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
19 protocols using ecis zθ instrument
1
Electric Cell-Substrate Impedance Sensing
2
Electric Cell-Substrate Impedance Sensing
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Electric cell-substrate impedance sensing (ECIS) measurements were performed using 8W1E + electrode arrays on an ECIS Zθ instrument (Applied Biophysics, Troy, NY). The measurements were performed as described previously [26 ]. A baseline was established using culture medium (400 μL well−1). The resistance was recorded in units of Ω at a frequency of 500 Hz. At 48 h after transfection, the cells were sub-cultured on an ECIS array. Exposure to hyperoxia or normoxia began when the electrode was covered with a monolayer of cells. The ECIS allows for a sensitive determination of the amount of current passing between cells and the resistance of the barrier (Rb) (in units of Ω cm2). Rb is a robust reporter of barrier function.
3
Wound-healing and ECIS-based Assays for Cell Migration
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Cell migration was assessed with a wound-healing assay. Cells were seeded into a 24-well plate at a density of 6 × 105 per well and then scratched to create a wound with a pipette tip after cells had formed a monolayer. The wound width was detected using a microscope at 0, 2, 4, 6, 8 and 10 h, and migration distances were measured using Image J software (www.ImageJ.net ).
In addition, an electric cell-substrate impedance sensing (ECIS)-Zθ instrument (Applied Biophysics Ltd.; Troy, NJ, USA) was used to monitor cell migration, as described previously [26 (link)]. Briefly, 6 × 104 cells, diluted in DMEM with 10% FCS, were seeded into each well of ECIS 96-plate arrays. Wounding was carried out by applying electric current (3000 μA, 60 kHz) once a confluent monolayer had formed. The migration data were collected continuously for 12 h.
In addition, an electric cell-substrate impedance sensing (ECIS)-Zθ instrument (Applied Biophysics Ltd.; Troy, NJ, USA) was used to monitor cell migration, as described previously [26 (link)]. Briefly, 6 × 104 cells, diluted in DMEM with 10% FCS, were seeded into each well of ECIS 96-plate arrays. Wounding was carried out by applying electric current (3000 μA, 60 kHz) once a confluent monolayer had formed. The migration data were collected continuously for 12 h.
4
Endothelial Monolayer Integrity Assay
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The electric cell-substrate impedance sensing (ECIS) method was used to measure the endothelial monolayer integrity. HBECs were cultured in 8-well electrode arrays (8W 10E, Applied BioPhysics Inc., Troy, NY, United States). After reaching confluence, cells were treated with different Hb species (Hb, metHb, and ferrylHb at a concentration of 50 μmol/L heme), and the complex impedance spectrum was monitored with an ECIS Zθ instrument (Applied BioPhysics Inc., Troy, NY, United States) for 4 h. Results are shown as the difference between monolayer resistance at 4,000 Hz at 0 time point and 4 h.
5
Real-Time Cell Migration Assay
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8W1E ECIS culturewares (Applied BioPhysics, Troy, NY) were used for migration assay as described previously (Tian et al., 2017 (link)). Briefly, 8 × 104 cells were seeded each well and cultured in incubator at 37°C in an atmosphere of 5% CO2. Media were changed next day; and cell monolayer were wounded using an elevated field pulse of 3,000 μA at 80,000 Hz applied for 20 s, producing a uniform circular lesion 250 μm in size. The wounds were tracked in real-time at 4,000 Hz using ECIS Zθ instrument (Applied BioPhysics, Troy, NY).
6
Cell Adhesion Dynamics with ECIS
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Cell–cell adhesion and cell–substrate adhesion were measured using ECIS. SKOV3ip cells with Rac1 OE or KD were plated at confluence on 96-well electrode arrays, 96W10idf (Applied BioPhysics). Electrodes were stabilized with l -cysteine. Treatment with l -cysteine enhances experimental repeatability between the ECIS wells (Applied BioPhysics). The cysteine displaces unwanted molecules that have been absorbed onto the gold electrodes and creates a hydrophilic substrate that helps with cell attachment and spreading over the electrodes (Applied BioPhysics). l -cysteine (10 mM) in water (Applied BioPhysics) was added to each well and incubated for 30 min at room temperature. l -Cysteine was removed, the wells were rinsed twice with water, and then the electrodes were washed with RPMI 1640 media without FBS. Media was removed and replaced with SKOV3ip cells in prewarmed complete RPMI growth media as detailed above. Electrode arrays were placed in an ECIS Zθ instrument (Applied BioPhysics) and resistance and capacitance were measured at 4000 and 64,000 Hz, respectively, every 10 min over 72 h. Each experiment included four replicates, and three independent biological replicates were completed. All values are expressed as mean ± SD.
7
ECIS-based Analysis of Cell Adhesion on Collagen
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The 8W10E arrays (IBIDI, Munich, Germany) were treated with 200 µL 10 mM L-cysteine dissolved in sterile water for 15 min at room temperature. Wells were washed 3× with 300 µL sterile water. Then, 200 µL PureCol Type 1 bovine collagen solution (Advanced Biomatrix, San Diego, CA, USA) diluted 1 in 100 in sterile water, was added to wells and incubated at 37°C overnight. Wells were washed 3× with BEGM. Then, 400 µL BEGM was added to each well and the array was stabilised in the electric cell-substrate impedance sensing (ECIS) Zθ instrument (Applied Biophysics, Troy, NY, USA). Medium was replaced with 7.5×104 cells in BEGM. The array was run at multiple frequencies (including the specific frequencies of 400 Hz and 32 kHz) for 72 h continuously on the ECIS Zθ instrument, as described previously [14 (link)].
8
Measuring Barrier Function of HEKa Cells
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The barrier function of HEKa cells was assessed by measuring the resistance of a cell‐covered electrode using an ECIS‐Zθ instrument (Applied Biophysics) as described previously.23 The change in TEER value after the addition of the test agent (0 h) was measured at a low frequency (4 kHz) for 48 consecutive hours.
9
Real-time TEER Monitoring of Ponatinib's Effect
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TEER values of HAEC monolayers treated with ponatinib was assessed in real-time by ECIS system using 8W10E+ array chambers. Briefly, the array chambers were treated with 10 mM L-Cysteine solution (room temperature, 15 min) followed by washing twice with ultra-pure water. The treated chambers were then coated with 0.2% gelatin type A. HAECs were seeded into the chambers and grown in complete ECM overnight to produce a confluent monolayer. Next day, complete ECM was replaced with low serum ECM (0.2% FBS, 1% P/S, no ECGF) and baseline resistance measurements were taken. Upon stabilization, ponatinib was added, and change in TEER values were recorded by an ECIS-Zθ instrument (Applied BioPhysics Inc., Troy, NY, USA) connected with a Dell personal computer equipped with ECIS software (Applied Biophysics). Figures illustrate normalized TERR values (where the value of 1.0 represents the basal TEER measurement immediately before adding ponatinib). Decrease in TEER indicates increased permeability (63 (link)).
10
Evaluating Endothelial Barrier Integrity via ECIS
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To measure endothelial monolayer integrity, we used Electric Cell-substrate Impedance Sensing (ECIS) method. Confluent HUVEC were pretreated in the presence or absence of DFC (50 µmol/L) for 16 h in medium containing 5% FBS. Then, cells were incubated in the presence or absence of TNF-α (1 ng/mL) for 6 h. The complex impedance spectrum was monitored with an ECIS Zθ instrument (Applied BioPhysics Inc., Troy, NY, USA) over 3 h. Intracellular gap formation was calculated based on the difference between monolayer resistance at 4000 Hz at 0-time points and 3 h.
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