Transepithelial electrical resistance was used to determine the integrity of brain endothelial monolayers using the Millicell Voltohmmeter (Millipore) with STX01 chopstick electrodes. In brief, bEnd.3 cells were inoculated in transwell inserts of 24-well plates at a density of 5 × 104/well. Prior to use, the machine was calibrated, then the longer electrode was placed so as to touch the bottom of the dish while the shorter electrode was prevented from reaching the bottom of the insert. The readings were corrected by transwell inserts with no cells (subtracted from each experimental measurement), then divided by filter size.
Millicell voltohmmeter
Manufactured by Merck Group
Sourced in United Kingdom
The Millicell Voltohmmeter is a compact, handheld device used to measure the electrical resistance and transepithelial/transendothelial electrical resistance (TEER) of cell monolayers grown on permeable membrane supports. It provides accurate measurements of the electrical properties of cell cultures, which are essential for evaluating cell barrier function and integrity.
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Lab products found in correlation
Human pedf elisa kit, by BioVendor (2 mentions)
Lsm 700 800 confocal microscope, by Zeiss (2 mentions)
Zen 2.3 sp1 black software, by Zeiss (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cell culture insert, by Thermo Fisher Scientific (1 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using millicell voltohmmeter
1
Brain Endothelial Cell Barrier Integrity
2
Transwell Nanoparticle Transport Assay
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The donor and receiver chambers of the transwell system faced the RBMECs and CTX TNA2, respectively. A nanoparticle concentration of 25 μg/mL in the ECM was added to the donor chamber and cultured in the humidified CO2 incubator at 37 °C for 4 h. The donor and receiver chambers were filled with the respective culture media to the same height. A Millicell voltohmmeter (Millipore) was applied to determine the TEER of the PET insert with and without the RBMECs/CTX TNA2.
3
Caco-2 Cell Permeability Assay Protocol
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Caco-2 cells were cultured in DMEM supplemented with 10% FBS, 1% NEAA, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. They were incubated at 37°C in a humidified atmosphere of 5% CO 2 in air. The monolayer became confluent 3 to 4 d after seeding at 1 × 10 6 cells/flask, and the cells were subcultured at split ratio of 1:5 by trypsinization (0.5% trypsin and 0.05% EDTA). Caco-2 cells used in this study were between passages 30 and 42.
For the permeability study, Caco-2 cells (1 × 10 6 cells/mL) were seeded onto cell culture inserts (0.4 μm pore size, 4.2-cm 2 growth surface, Nunc, Thermo Scientific) in a 6-well culture plate. The medium was changed every other day for at least 21 d until the Caco-2 cells were fully differentiated. The integrity of the cell layer was evaluated by measuring the transepithelial electrical resistance (TEER) by using a Millicell Voltohmmeter (Millipore Corp., Bedford, MA). Only Caco-2 monolayers showing TEER >400 Ω•cm 2 were used for permeability experiments (Miguel et al., 2008) . The integrity of the monolayers was checked before and after the experiment.
For the permeability study, Caco-2 cells (1 × 10 6 cells/mL) were seeded onto cell culture inserts (0.4 μm pore size, 4.2-cm 2 growth surface, Nunc, Thermo Scientific) in a 6-well culture plate. The medium was changed every other day for at least 21 d until the Caco-2 cells were fully differentiated. The integrity of the cell layer was evaluated by measuring the transepithelial electrical resistance (TEER) by using a Millicell Voltohmmeter (Millipore Corp., Bedford, MA). Only Caco-2 monolayers showing TEER >400 Ω•cm 2 were used for permeability experiments (Miguel et al., 2008) . The integrity of the monolayers was checked before and after the experiment.
4
Characterization of Human ESC-Derived RPE
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Human ESC-RPE authentication was performed as previously described [16 (link)]. Briefly, transepithelial electrical resistance (TEER) was triplicate measured with Millicell volt-ohm meter (Merck Millipore) [17 (link)]. The key RPE protein expression and localization were verified with indirect immunofluorescence labeling for zonula occludens-1 (ZO-1), claudin-3, claudin-19, sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), bestrophin, and MER Proto-Oncogene, tyrosine Kinase (MERTK). Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) was carried out from apical and basal media collected after overnight incubation and analyzed with the Human PEDF ELISA kit (BioVendor) following the manufacturer’s instructions. Phagocytosis assay was conducted with porcine photoreceptor outer segments (POS) by 4 h apical incubation at 37 °C in the presence of 10% fetal bovine serum (Thermo Fisher Scientific), followed by labeling with anti-rhodopsin antibody and tetramethylrhodamine (TRITC). The nuclei were counterstained with DAPI included in ProLong Gold mounting medium (Thermo Fisher Scientific). Images were acquired with an LSM 700-800 Confocal microscope (Carl Zeiss) and processed with the Zen 2.3 SP1 Black software (Carl Zeiss). All primary and secondary antibody details appear in Table S1 (Additional file 1 ).
5
Caco-2/TC7 Monolayer Permeability Assay
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Caco-2/TC7 cells were cultured on 12-well transwell plates for 21 days, washed three times with warm transport buffer (20 mM HEPES buffer, 5.4 mM potassium chloride, 137 mM sodium chloride, 2.4 mM calcium chloride and 26.8 mM of sodium bicarbonate at pH 7.4) and incubated at 37 °C, 10% CO2 in transport buffer for 30 min. The trans-epithelial electric resistance (TEER) was measured using a Millipore Millicell Voltohmmeter (Merck Millipore, UK) at three places per well. TEER values greater than 200 MΩ indicated tight Caco-2/TC7 monolayer formation. For transport experiments, 1 mL of transport buffer was added to the basolateral side and 0.5 mL of transport solution containing glucose or sucrose, at concentrations stated in the figure legends, was added to the apical side. Samples were incubated for the specified time at 37 °C, 10% CO2. The apical and basolateral solutions were then transferred to microcentrifuge tubes and stored at −80 °C until analysis. During the transport assay, OLE was absent, irrespective of whether the cells had been previously treated with OLE or control.
6
Characterization of Human ESC-RPE Cells
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Human ESC-RPE authentication was performed as previously described [15] . Brie y, transepithelial electrical resistance (TEER) was triplicate measured with Millicell volt-ohm meter (Merck Millipore) [16] . Key RPE protein expression and localization was veri ed with indirect immuno uorescence labeling for zonula occludens-1 (ZO-1), claudin-3, claudin-19, sodium-potassium adenosine triphosphatase (Na + /K + -ATPase), bestrophin, and MER Proto-Oncogene, tyrosine Kinase (MERTK). Enzyme-linked immunoassay (ELISA) for pigment epithelium-derived factor (PEDF) was carried out from apical and basal media collected after overnight incubation and analyzed with the Human PEDF ELISA kit (BioVendor) following manufacturer's instructions. Phagocytosis assay was conducted with porcine photoreceptor outer segments (POS) by 4 hours apical incubation at 37°C in the presence of 10% fetal bovine serum (Thermo Fisher Scienti c), followed by labeling with anti-rhodopsin antibody and tetramethylrhodamine (TRITC). Nuclei were counterstained with DAPI included in ProLong Gold mounting medium (Thermo Fisher Scienti c). Images were acquired with an LSM 700-800 Confocal microscope (Carl Zeiss) and processed with the Zen 2.3 SP1 Black software (Carl Zeiss). All primary and secondary antibody details appear in Table S1 (Additional le 1).
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