Cells were cultured on 12-Transwell permeable polycarbonate filters (3-µm pore size, Corning). The Trans Epithelial Electric Resistant (TEER) across the monolayer was measured using an epithelial tissue Volt-Ohmmeter (Millipore) every 3–4 days, and the cells were used when a plateau was reached (approximately 500 Ω/cm2 Caco-2, approximately 300 Ω/cm2 IPEC-J2). Bacteria were added to the upper chamber at MOI 50 and coincubated with EICs for 6 hours. At each 2-hourly sampling, medium in the lower chamber was replaced to prevent growth of translocated bacteria. Samples of the upper and lower chamber were taken every 2 hours and plated. We calculated the percentage bacterial translocation by dividing the CFUs number in the lower chamber by the CFUs number in the upper chamber. Data are given as mean ± SEM of at least 3 independent experiments, performed in triplicates by 2 different operators. SS2 BM407 was included in each experiment. Low, medium, and high translocation were defined as 0%–0.2%, 0.2%–1.0%, and ≥1.0% of starting inoculum, respectively.
Volt ohm meter
Manufactured by Merck Group
Sourced in France
A volt-ohm meter is an electronic measuring instrument that can be used to measure voltage, current, and resistance in electrical circuits. It is a versatile tool that allows users to assess the electrical characteristics of various components and systems.
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Lab products found in correlation
Fluostar omega, by BMG Labtech (2 mentions)
Transwell filter chamber, by Corning (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Flagellin, by Thermo Fisher Scientific (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Transwell insert, by Corning (1 mentions)
Transwell inserts with 0.4 μm pore polycarbonate membranes, by Corning (1 mentions)
Collagen 4, by Merck Group (1 mentions)
Endohm 12 chamber, by World Precision Instruments (1 mentions)
Millicell pcf, by Merck Group (1 mentions)
8 protocols using volt ohm meter
1
Bacterial Translocation Quantification
2
Assessing Epithelial Permeability
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To assess paracellular permeability, FITC-labeled dextran 4 kDa (1 mg/mL; TdB Consultancy AB, Uppsala, Sweden) or FITC-sulfonic acid 0.4 kDa (0.2 mg/mL; Thermofisher Scientific) was added to the apical medium. Samples of basal medium were collected after 4 h, and fluorescence was determined with a microplate fluorometer (FLUOstar Omega; BMG Labtech). Transepithelial electrical resistance (TEER), which is inversely proportional to permeability to ions, was measured after treatment using a volt-ohm meter (Millipore, Guyancourt, France).
3
Wnt Signaling Pathway Analysis in Cell Lines
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MDCK II, HEK293, and L cells, all of which were kindly provided by Dr. M. Takeichi, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or in a 1:1 mixture of DMEM and Ham’s F-12 medium supplemented with 8% fetal calf serum (FCS) and antibiotics. Polarized MDCK II cell cultures were established by plating the cells at 2 × 106 cells in 100-mm Transwell filter chambers (Corning), and the cells were incubated for 7 days with a daily change of fresh culture medium. The integrity of the monolayer was verified by measuring the Trans-epithelial Electrical Resistance with a volt-ohm meter (Millipore). MDCK II and HEK293 cells stably expressing wild-type mouse Wnt3a, mutant Wnt3a (Wnt3a (S209A)) or wild-type mouse Wnt11 were established by transfection with wild-type Wnt3a, Wnt11 or Wnt3a (S209A) plasmid as indicated previously50 (link). In these cells, Wnt genes were expressed under the control of the CMV promoter. Cells were selected and maintained in culture medium containing 400 μg/mL and 200 μg/mL G418, respectively. L cells stably expressing wild-type Wnt3a were established as previously described41 (link). The supernatant from cultures of Wnt3a- or Wnt11-producing cells was prepared as previously described50 (link).
4
Inflammatory Response in Caco-2 Cells
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Bacteria were added in the apical compartment of the Caco-2 cells inserts. Basolateral compartment of the inserts were filled with DMEM without FCS or antibiotics. Inserts were then incubated for 16 h at 37°C in a 5% CO2 incubator when pro-inflammatory signals were applied. Deoxynivalenol (DON, 10 μM final concentration, obtained from Romer lab) was added to the apical compartment, while human recombinant IL-1β (20 ng/ml final concentration, obtained from Peprotech), or flagellin (1 μg/ml final concentration, extracted from Salmonella typhimurium, obtained from Invivogen) were added basolaterally to Caco-2. Epigallocatechin gallate (EGCG, 10 μM final concentration, obtained from Sigma Aldrich) was added to the apical compartment and used as positive anti-inflammatory control. After 6 h at 37°C, basolateral media were collected and stored at −80°C before IL-8 cytokine measurement by ELISA (BD Biosciences) was performed. TEER was measured using a Volt/Ohm meter (Millipore).
5
Evaluating Endothelial Barrier Integrity
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Cultivation of cell monolayers on the upper side of the Transwell insert (Corning, United States) at 37°C, 5% CO2. In the upper and lower chambers, 200 μL and 600 µL culture medium were distributed, respectively. After treatment, the Volt-Ohm Meter (Millipore, United States) was used to measure TEER following the protocol. The values (Ω cm2) of TEER were expressed by subtracting the resistance of the blank insert and correcting for the surface area.
Culture HUVECs as previously described. After treatment, FITC-dextran (1 mg/ml, Sigma-Aldrich, United States) was added to the upper chambers and cultured for 45 min 100 µL samples were collected from the upper and lower chambers for fluorescence. The dextran permeability coefficient (Pd) was used to evaluate the permeability of the endothelial monolayer.
[A] is the dextran concentration of the lower chamber, t is time, A is the membrane area; V is the volume of the lower chamber [L] is the dextran concentration of the upper chamber (Wu et al., 2020 (link)).
Culture HUVECs as previously described. After treatment, FITC-dextran (1 mg/ml, Sigma-Aldrich, United States) was added to the upper chambers and cultured for 45 min 100 µL samples were collected from the upper and lower chambers for fluorescence. The dextran permeability coefficient (Pd) was used to evaluate the permeability of the endothelial monolayer.
[A] is the dextran concentration of the lower chamber, t is time, A is the membrane area; V is the volume of the lower chamber [L] is the dextran concentration of the upper chamber (Wu et al., 2020 (link)).
6
Measuring Transepithelial Electrical Resistance
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For the measurement of the transepithelial electrical resistance (TEER), 12 mm Transwell® inserts with 0.4 μm pore polycarbonate membranes (Corning) coated with collagen IV (20 μg/ml, Sigma-Aldrich) were used. OKG4 cells (2.7 × 104) in 500 μl in DermaLife K medium containing 60 μM Ca2+ were seeded to the filter, and the medium was changed every 1–3 days. When cells reached confluence (approx. after 7 days), they were cultured in a medium containing 1.4 mM Ca2+ to induce terminal cell differentiation [29 (link)] and the formation of tight junctions [30 (link)]. Subsequently, 50 μg/ml of the nanocarrier solution was applied to the cells. As a control, the TEER of cells that were kept in DermaLife K medium containing 60 μM Ca2+ was determined. The TEER values were measured using an Endohm-12 chamber (World Precision Instruments) and a volt-ohm-meter (Millipore). After subtracting the blank filter’s TEER, the value was multiplied by the filter area (1.12 cm2). Four independent experiments were performed in duplicate.
7
Differentiation of CuFi-8 cells into Air-Liquid Interface Culture
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CuFi-8 cells differentiate on collagen-coated, semipermeable membrane inserts (0·6 cm2, Millicell-PCF; Millipore, Billerica, MA) into an air-liquid interface culture. After 3–4 weeks of culture in DMEM:F12 medium (50%:50%) containing 2% Ultroser G (Pall BioSepra, Cergy-Staint-Christophe, France) the polarity of the HAE was determined based on the transepithelial electrical resistance (TEER) using an epithelial Volt-Ohm Meter (Millipore). HBoV1 infection was performed at minimal values of 600–800 Ω per insert [27 (link)].
8
Evaluating Paracellular Permeability
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To assess paracellular permeability, 1 mg/ml of 4 kDa FITC-dextran (TdB Consultancy AB, Uppsala, Sweden) was added to the apical medium on the last day of treatment. Samples of basal medium were collected after 4 h, and fluorescence was determined with a microplate fluorometer (FLUOstar Omega; BMG Labtech, Champigny s/Marne, France). Transepithelial electrical resistance (TEER), which is inversely proportional to ion permeability, was measured before and after treatments using a volt-ohm meter (Millipore, Guyancourt, France).
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