Cells (3 × 105/well) were seeded in the upper room of Transwell plates (Corning, USA), then 2.6 ml medium was added into the basal chamber. Millicell-ERS instrument (Millipore, Bedford, USA) was used to measure TEER values. The measured values in the blank wells of uninoculated cells were background TEER. The final TEER values were corrected for background and displayed as Ω cm2.
Millicell ers instrument
Manufactured by Merck Group
Sourced in United States
The Millicell-ERS instrument is a compact and easy-to-use device designed for measuring the electrical resistance of cell monolayers in cell culture. It provides a reliable and accurate way to assess the integrity and barrier function of cell layers.
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10 protocols using millicell ers instrument
1
Transwell Assay for TEER Measurement
2
Anti-Inflammatory Effects of SQE in Caco-2/RAW 264.7 Co-Culture
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A co-culture system was established as described previously [21 (link)]. Briefly, Caco-2 cells (passage numbers: 48 - 62) were seeded onto Transwell insert plates (3.75 × 105 cells/well; Corning Costar Corp., Cambridge, MA, USA) and incubated for 21 d to obtain an integrated cell monolayer with a transepithelial electrical resistance value (TER) 1,200 Ω cm2 using a Millicell-ERS instrument (Millipore Corporation, Billerica, MA, USA). Cell culture medium was changed every 3 d. RAW 264.7 macrophages (passage numbers: 10 - 30) were seeded in 6-well tissue culture plates (8.5 × 105 cells/well) and incubated overnight. After replacing the media with RPMI1640, transwell inserts containing Caco-2 cells were added to the 6-well plates containing RAW 264.7 macrophages (Fig. 1 ). To evaluate the anti-inflammatory effects of SQE in this co-culture system, various concentrations of SQE were applied to the apical side of the transwell insert for 3 h, followed by the addition of 1 µg/ml lipopolysaccharide (LPS, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to the basolateral side. After an additional incubation overnight, culture supernatants from the basolateral side were collected to measure levels of nitric oxide (NO), PGE2, IL-6, and IL-1β. The cultured cells were subsequently harvested to isolate total RNA for RT-PCR and western blot analyses.
3
Transepithelial Electrical Resistance and Permeability Assays
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After BTB models were established, the TEER assays were performed by millicell-ERS instrument (Millipore, Billerica, MA, United States). Based on our previous experiments (Cai et al., 2015a (link)), in 96 h of co-culture, TEER values were measured. Before final resistances were calculated, background electrical resistances had been subtracted. The final TEER values were measured as ohms per square centimeter. After BTB models were constructed, 1 ml serum-free EBM-2 containing 0.5 μmol/L HRP was added into the upper compartment of the transwell. In 1 h, the medium in the lower compartment was collected and TMB colorimetry was applied to test the collected samples by spectrophotometer at 370 nm. The HRP flux was shown as picomoles passed per square centimeter surface area per hour (pmol/cm2/h).
4
Evaluating Gel Formulation Epithelial Integrity
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The effect of the gel formulation on its ability to maintain an intact epithelium was determined by measuring transepithelial resistance (TER) using Milli cell–ERS (electrical resistance system) voltmeter [21 (link)]. Caco-2 cells (5 × 105 cells/well) were grown in transwells (Corning Incorporated, Costar) to form tight junctions and culture medium was dispensed in the basolateral compartment of each well. Apical and basolateral media were replaced, and TER was measured daily with Millicell-ERS instrument (Millipore Corporation, Billerica, MA, USA). When plateau TER was reached, gel formulation/gel base (5 mg/mL) was added in the culture medium and cells were further incubated in humidified atmosphere of 5% CO2 at 37 °C. TER was measured at 0 min and 1, 2, 4, 8, and 24 h. Triton X-100 (0.1%) was used as positive control.
5
Measuring TEER of Brain Microvascular Endothelial Cells
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A Millicell-ERS instrument (Millipore, MA, USA) was used to detect the TEER values of BMECs. In detail, the isolated primary astrocytes (1 × 104 cells) were grown on six-well plates for 2 days. Then, the coculture inserts were placed at room temperature for 25 min. Subsequently, we detected the values of TEER immediately after changing the medium. Before calculating the final resistance, the background resistance was subtracted and the TEER value was expressed as Ω cm2.
6
Evaluating Intestinal Barrier Integrity
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Next, to evaluate the intestinal epithelial barrier function, Caco-2 cells were plated into the upper chamber of the Transwell system (0.4 μm pore, 3460, Corning, NY). Epithelial permeability was assessed by the analysis of transepithelial electrical resistance (TEER) and the paracellular flux of FITC-dextran (FD4, Sigma-Aldrich). The electrical resistance of the Caco-2 cell monolayers cultured in the Transwell chamber was assessed using a Millicell-ERS instrument (Millipore, Bedford, MA). FITC-dextran was added to the upper chamber at a final concentration of 1 mg/ml. Two hours after the addition of FITC-dextran, the medium from the lower chamber was collected, and fluorescence intensity was measured using a fluorescence microplate reader (Varioskan Flash 3001, Thermo Fisher Scientific).
7
Measuring Endothelial Cell Barrier Integrity
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The bEnd.3 cells were seeded in an insert with 0.4 μm pore (Millipore, USA) and allowed to reach a confluent state. Before every measurement, cells were preequilibrated with HBSS for 30 min and resistance was measured using a Millicell® ERS instrument (Millipore, USA) according to the manufacturer's instructions. TEER values were calculated using the formula: TEER (Ω•cm [2 (link)]) = [TEER total - TEER blank] × membrane area.
8
Measuring Caco-2 Cell Monolayer Integrity
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Caco-2 cells with different treatments were seeded on polycarbonate 12-well Transwell filter inserts (Corning Costar Corp., Cambri dge, MA, USA; pore size 0.4 μm, growth area 1.12 cm2) with 2 × 105 cells per well. Before TEER measurement, Caco-2 cells on each well were grown for 21 days and formed a polarized monolayer. The Millicell ERS instrument (Millipore, Bedford, MA, USA) was used to determine the TEER values of the Caco-2 cell monolayer. The changes of TEER were calculated with the formula: TEER = (R1-R0) × A (Ω · cm2). R1 and R0 represented the TEER values of the cell monolayer wells and the background wells without cells respectively, and A (cm2) represented the surface area of cell monolayer on the insert.
9
Evaluating Intestinal Barrier Function
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TJ barrier function was measured by determining transepithelial electrical resistance (TEER) in differentiated Caco-2 monolayers. CWE (50 or 100 μg/mL), CA (0.35 μg/mL), and lipopolysaccharide (LPS) (10 μg/mL) were injected into the apical well and incubated overnight with an inflammatory cytokine cocktail in the basal well (IFN-γ and TNF-α, 50 ng/mL; IL-1β, 25 ng/mL; LPS 10 ng/mL). The TEER values were measured three consecutive times 48 h after treatment with the cytokine cocktail using a Millicell® ERS instrument (Millipore, Bedford, MA, USA). The data are presented as a % of the initial value.
10
Measuring BTB Integrity via TEER
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A Millicell-ERS instrument (Millipore, Billerica, MA, USA) was used to measure the integrity of the BTB after in vitro BTB models were established. Before measuring the TEER values, co-culture inserts were placed in room temperature for 30 min. TEER values were measured directly after the medium exchange. Then, the background electrical resistance was subtracted before final resistances were calculated (Ω·cm2).
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