Caco-2 cells were seeded on tissue culture polycarbonate membrane filters (pore size, 0.4 μm) on 24 well transwell plates (SPL, Gyeonggi, Korea) at a seeding density of 5 × 104 cells/cm2. Culture medium was added to the upper and lower chambers and changed every second day. The cells were allowed to differentiate for 14 days after seeding. Prior to TEER measurement, the standard medium was substituted with media containing 0.01 μg/mL or 0.1 μg/mL LPS with or without 1 μg/mL Antarctic algae extract. The electrical resistance was measured using a Millicell ERS meter (Millipore, Bedford, MA, USA) and calculated as a percent change of Ω cm2 compared with the control group.
Millicell ers meter
Manufactured by Merck Group
Sourced in United States
The Millicell ERS meter is a laboratory equipment used to measure the electrical resistance and impedance of cell cultures and tissues. It provides accurate measurements of the integrity and permeability of cell monolayers or tissues. The device is designed for use in cell biology, tissue engineering, and related research applications.
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14 protocols using millicell ers meter
1
Caco-2 Barrier Function Modulation
2
Endothelial Monolayer Characterization for Amyloid and Inflammation Studies
3
Evaluating Barrier Function Efficiency
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The efficiency of the barrier functions was evaluated by measuring TEER using a voltmeter [29 (link)].
Caco-2 cells were placed in Transwell polyester membrane cell culture inserts (transparent PET membrane: 0.4 μm pore size; BD Falcon) as previously described [29 (link)]. Culture media was replaced every day.
The integrity of the cell monolayers were monitored by measuring the trans-epithelial electric resistance of the monolayer at confluence from day 14° to day 20° since cells were seeded. When a stable value was reached, a pre-treatment of 24 h was done adding BOS (1 μg/mL) and CUR (1 μg/mL) at the apical chamber in the appropriate wells.
TEER measurements were performed in HBSS (Hanks’ Balanced Salt solution, Lonza, Milan, Italy) after an equilibration period at room temperature [87 (link),88 (link)]. Only cells with TEER value within 360–500 Ω × cm2 were used for the experiments [89 (link),90 (link),91 (link)].
Treatments were added to the apical chamber and inflammatory stimulus (LPS 500 ng/mL) to the basal chamber. Millicell® ERS meter, Millipore Corporation (Bedford, MA, USA) connected to a pair of chopstick electrodes was inserted in the donor and receiver chambers and the TEER variation at 3, 6, 21 and 24 h after the stimulation was recorded.
TEER was expressed as percentage of resistance, normalized to initial value.
Caco-2 cells were placed in Transwell polyester membrane cell culture inserts (transparent PET membrane: 0.4 μm pore size; BD Falcon) as previously described [29 (link)]. Culture media was replaced every day.
The integrity of the cell monolayers were monitored by measuring the trans-epithelial electric resistance of the monolayer at confluence from day 14° to day 20° since cells were seeded. When a stable value was reached, a pre-treatment of 24 h was done adding BOS (1 μg/mL) and CUR (1 μg/mL) at the apical chamber in the appropriate wells.
TEER measurements were performed in HBSS (Hanks’ Balanced Salt solution, Lonza, Milan, Italy) after an equilibration period at room temperature [87 (link),88 (link)]. Only cells with TEER value within 360–500 Ω × cm2 were used for the experiments [89 (link),90 (link),91 (link)].
Treatments were added to the apical chamber and inflammatory stimulus (LPS 500 ng/mL) to the basal chamber. Millicell® ERS meter, Millipore Corporation (Bedford, MA, USA) connected to a pair of chopstick electrodes was inserted in the donor and receiver chambers and the TEER variation at 3, 6, 21 and 24 h after the stimulation was recorded.
TEER was expressed as percentage of resistance, normalized to initial value.
4
Measuring Cell Permeability and TEER
5
Transepithelial Electrical Resistance of Caco-2 Cells
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Caco-2 cells were cultured in Transwell® 24-well plates (Corning Costar®) on insert membranes, with a surface area of 0.33 cm2 and pore size of 0.4 μm, to form confluent monolayers. The positive control consisted of 0.5% w/v TMC (a known tight junction modulator), while the test solutions consisted of A. vera gel and whole-leaf extract, each in four different concentrations ranging from 0.1 % w/v to 1.5% w/v. Serum-free DMEM alone was used as the negative control.
The TEER measurements of the Caco-2 cell monolayers on insert membranes in 24-well Transwell® plates commenced one hour prior to addition of the test solutions, to obtain the TEER values, at a baseline level. DMEM buffered with HEPES (pH = 7.4) (1 mL) was added to the basolateral chamber and incubated for 30 min, prior to the addition of the test solutions (200 µL) to the apical chamber on top of the cell monolayers, on the filter membranes. The TEER (T0) was measured directly, after application of the test solutions to the apical chamber. TEER measurements were then taken at 20 min intervals up to 120 min, after addition of test solutions. TEER was measured with a Millicell ERS meter (Millipore, Billerica, MA, USA) that was connected to a set of chopstick electrodes.
The TEER measurements of the Caco-2 cell monolayers on insert membranes in 24-well Transwell® plates commenced one hour prior to addition of the test solutions, to obtain the TEER values, at a baseline level. DMEM buffered with HEPES (pH = 7.4) (1 mL) was added to the basolateral chamber and incubated for 30 min, prior to the addition of the test solutions (200 µL) to the apical chamber on top of the cell monolayers, on the filter membranes. The TEER (T0) was measured directly, after application of the test solutions to the apical chamber. TEER measurements were then taken at 20 min intervals up to 120 min, after addition of test solutions. TEER was measured with a Millicell ERS meter (Millipore, Billerica, MA, USA) that was connected to a set of chopstick electrodes.
6
Transepithelial Electrical Resistance Assay
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Transepithelial electrical resistance (TEER) was assayed using a MilliCell-ERS-meter (Millipore, Molsheim, France). TEER was measured before and after 1, 2, 3, 4 and 24 h of exposure of Caco-2 cells to each sample (P, Pc, A, Ac, T and Tc). Inserts with Caco-2 cells showing TEER values < 800 Ω·cm2 before starting the experiments were excluded. All measurements were carried out at 37 °C in order to reduce the influence of temperature changes on cells and, then, on TEER values. Three biological replicates were used.
7
Transepithelial Electrical Resistance Assay
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A transepithelial electrical resistance (TEER) assay was performed as reported previously 27 . Briefly, Caco‐2 cells were seeded on tissue culture polycarbonate membrane filters (pore size, 0.4 μm) in 24‐well transwell plates at a seeding density of 2 × 105 cells/cm2 (Corning, Inc., Acton, MA, USA). The culture medium was added to the upper and lower chambers and was changed every second day. The cells were maintained to differentiate for 14 days after seeding with monitoring of TEER. Prior to TEER measurement, the standard medium was substituted with foetal bovine serum‐free culture medium. The electrical resistance was measured using a Millicell ERS meter (Millipore, Bedford, MA, USA) and calculated as Ω cm2.
8
Caco-2 Monolayer Permeability Assay
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Caco-2 cells (15 × 104) were seeded on Transwell™ polyester membrane cell culture inserts (transparent PET membrane: 1.0 cm2 growth surface area, 0.4 μm pore size; BD Falcon™) in 24-well plates and incubated with DMEM at 37°C in a humidified atmosphere and 5% CO2. Culture media was replaced every two days until confluent monolayer was obtained. The integrity of the cell monolayers was monitored by measuring the transepithelial electric resistance (TEER) from day 14th to day 21st after seeding. A 24 h pre-treatment was done adding CBD or THC (0.01–0.1 ug/mL) in the apical chamber. The TEER assay was performed in Hanks’ Balanced Salt solution (HBSS, Cambrex Lonza) with 10 mM Hepes and 10 mM d -glucose (pH = 7.4), after an equilibration period at RT (Liu et al., 2010 (link)). Treatments were added to the apical chamber and inflammatory stimuli to the basal chamber. Millicell® ERS meter (Millipore Corporation) connected to a pair of chopstick electrodes were inserted in the donor and receiver chambers and the 24 h-time courses of TEER variation was recorded (1-3-6-21-24 h). TEER was expressed as percentage of resistance, normalized to initial value (Governa et al., 2018 (link)).
9
Measuring Caco-2 Cell Monolayer Permeability
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TEER measurements, used to measure the cell monolayers permeability (19 (link)), were determined using millicell® ERS meter (Millipore Corporation) connected to a pair of chopstick electrodes, according to Srinivasan et al. (20 (link)). Caco-2 cells were treated for increasing time intervals as detailed above. At each time point, TEER was recorded. The values were expressed as percent of resistance and normalized to the initial value.
10
Evaluating Intestinal Barrier Integrity
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The efficiency of the intestinal barrier functions was evaluated by measuring trans-epithelial electric resistance (TEER) using a voltmeter [43] (link). Caco-2 cells (8 × 10 5 ) were placed in transparent polyester membrane cell culture inserts with 0.4 μm pore size (Sarstedt), and cultured in 24-well plates, as previously described [44] (link). Culture medium was replaced every other day. The integrity of the cell monolayers was monitored by measuring the TEER of the monolayer from day 14th to day 21st after seeding. When a stable value was reached, a 12 h pre-treatment was done by adding pre-and post-digested samples (diluted 1:1000) in the apical chamber in appropriate wells and TEER was measured after 0 and 12 h. Then, 0.5 mM H 2 O 2 was added to the basal chamber and TEER was measured after 0, 4, 8, and 24 h. TEER measurements were performed in HBSS with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mM d-glucose (pH = 7.4), after an equilibration period at rt, using a Millicell ® ERS meter, (Millipore Corporation, Bedford, MA) connected to a pair of chopstick electrodes. Only cells with TEER value between 360 and 500 Ω cm 2 were used for the experiments [45, (link)46] (link). Treatments were performed in duplicate in three independent experiments and TEER was expressed as percentage of resistance, normalized to initial value.
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