To evaluate the putative effects of serum from acutely YFV-infected patients on endothelial barrier function, we used the TEER assay as described previously (26 (link), 27 (link)). In brief, human umbilical vein endothelial cells, kindly donated by Dr. Miriam Fonseca-Alaniz (Instituto do Coração, InCor, University of São Paulo, Brazil) were seeded (6×104 cells/well) in Transwell polycarbonate membrane inserts (0.4 μm pore, 6.5 mm diameter; Corning Inc.) in endothelial cell growth basal medium 2 supplemented with an Endothelial Cell Growth Medium-2 (EGM-2TM) supplemental bullet kit (Lonza). After 72h of incubation at 37°C and 5% CO2, cells were treated with human sera (10% final vol/vol concentration) obtained from YFV-positive severe and non-severe patients or YFV-negative blood donors (healthy controls). TEER values, expressed in Ohms (Ω), were collected at sequential 2-h time-points 2–10h following treatments using an Epithelial Volt Ohm Meter (EVOM) with a “chopstick” electrode (World Precision Instruments). Resistance of inserts with no cells (blank) and inserts with cells (untreated) containing medium alone, were used to calculate relative TEER as a ratio of the corrected resistance values as (Ω experimental condition - Ω blank)/(Ω untreated - Ω blank). Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as positive control.
Chopstick electrode
Manufactured by World Precision Instruments
Sourced in United States, United Kingdom
Chopstick electrodes are laboratory equipment designed for a variety of applications. They are made of inert materials and feature a unique shape that allows for precise positioning and manipulation. The core function of chopstick electrodes is to provide a means for making electrical contact with samples or materials under investigation.
Automatically generated - may contain errors
Lab products found in correlation
Epithelial voltohmmeter, by World Precision Instruments (4 mentions)
Evom epithelial volthometer instrument, by World Precision Instruments (2 mentions)
Evom2 epithelial voltohmmeter, by World Precision Instruments (2 mentions)
Evom epithelial voltohmmeter, by World Precision Instruments (2 mentions)
Evom meter, by World Precision Instruments (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Transwell polycarbonate membrane insert, by Corning (1 mentions)
Endothelial cell growth medium 2 egm 2tm supplemental bullet kit, by Lonza (1 mentions)
Cyclosporin a, by Merck Group (1 mentions)
Ko143, by Merck Group (1 mentions)
Mk571, by Merck Group (1 mentions)
Evom2 system, by World Precision Instruments (1 mentions)
Nunc lab tek 2, by Thermo Fisher Scientific (1 mentions)
Its premix universal culture supplement, by Corning (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fbs dmem f 12 medium, by Thermo Fisher Scientific (1 mentions)
Voltohmmeter, by World Precision Instruments (1 mentions)
Transwell polycarbonate membrane system, by Corning (1 mentions)
Mem without phenol red, by Thermo Fisher Scientific (1 mentions)
22 protocols using chopstick electrode
1
Evaluating Endothelial Barrier Function in Yellow Fever
2
Intestinal Permeability Co-Culture Model
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The intestinal model, composed of a co-culture of Caco-2 and HT29-MTX cell lines, was prepared according to the original model developed and validated by Araújo and Sarmento, and slight modified by González et al. [29 (link)]. The experiments were performed 21 days after seeding the cells in 12-well plates. During this period, the transepithelial electrical resistance (TEER) was monitored to evaluate the cell monolayer integrity. On the last day, cell monolayers were pre-equilibrated with fresh HBSS (pH 7.4) at 37 °C for 30 min. Afterwards, 0.5 mL of the sample (500 µg/mL) prepared in HBSS was added to the apical side of the co-culture monolayers and 1.5 mL of HBSS to the basolateral side. Samples were withdrawn from the receptor side at different timepoints (0, 15, 30, 45, 60, 90, 120, 150, 180, and 240 min) to determine the bioactive compounds transported across the monolayer. At the same time, the TEER was evaluated, employing an EVOM Epithelial Volthometer Instrument equipped with a chopstick electrode (World Precision Instruments, Sarasota, FL, USA). After each sampling time, the basolateral side was replaced with the same HBSS volume. Samples were conserved at −20 °C for subsequent LC/ESI-MS analysis, according to Section 4.5 . The transepithelial electrical resistance (TEER) was quantified before, during, and at the end of the assay.
3
Transwell Permeability and Efflux Assays
4
Intestinal Permeability Evaluation in Caco-2/HT29-MTX Co-Culture
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The intestinal permeability was carried out in a co-culture model with Caco-2 and HT29-MTX [40 (link)]. The extract was added to the apical side of the model. A sample from the basolateral side was collected at different timepoints (0, 15, 30, 45, 60, 90, 120, 150, 180, and 240 min). Samples were analyzed by HPLC-MS assay (Section 3.5 ). The TEER of the model was evaluated before, during and at the end of the permeability assay using an EVOM Epithelial Volthometer Instrument equipped with a chopstick electrode (World Precision Instruments, Sarasota, FL, USA).
5
Measuring Epithelial Barrier Integrity
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Tissue permeability was assessed at T12 and PT20 by measuring changes in trans-epithelial electrical resistance (TEER) using an EVOM2 epithelial volt-ohmmeter and a chopstick electrode (World Precision Instruments, Sarasota, FL). Prior to the TEER measurement, ALI cultures were briefly washed twice with DPBS from the apical side to remove the excess mucus. Cultures were then equilibrated to room temperature in fresh Maintenance Medium (400 µL per well). DPBS (200 µL) was added to the apical chamber of the culture inserts. Measurements on each culture were made at 3 positions that were 120° apart and the average was used for data analysis.
6
Caco-2 Cell Monolayer Differentiation
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A total of 82,000 cells/cm 2 (Caco-2/wt and Caco-2/hPXR) or 79,000 cells/cm 2 (Caco-2/hCAR) were seeded onto uncoated polycarbonate filters (Transwell ® , pore size 0.4 mm, Corning, NY) and grown for 21 days. Standard growth medium (with volumes according to manufacturer's instructions) was changed on the day of the seeding, thereafter 3 times per week and 1 day before the experiments. VD3-treated cells were obtained from wild-type or modified cells lines by supplementing the standard growth medium with 250 nM 1a,25-dihydroxyvitamin D 3 after 8 days of culture on Transwell ® filters 20 and were used for CYP3A4 metabolism studies. Transepithelial electrical resistance (TEER) of filtergrown cells was measured prior the experiments with an epithelial voltohmmeter and a chopstick electrode (World Precision Instruments, Sarasota, FL).
7
IPEC-J2 Cell Culture and Infection
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Nontransformed porcine jejunal epithelial cells (IPEC-J2) were grown in co-culture media that included Dulbecco’s minimal essential medium:nutrient mixture F-12 (with L-glutamine and 15 mmol/L HEPES, catalog 10-092-CM; Corning, Corning, NY) supplemented with 5 μg/mL each of insulin, transferrin, and selenium (ITS Premix universal culture supplement, catalog 354350; Corning), epidermal growth factor (5 ng/mL, catalog 354052; Corning), penicillin (50,000 IU/mL), streptomycin (50,000 mg/mL) (100× penicillin-streptomycin solution, catalog 30002CI; Corning), and 5% porcine serum (catalog 26250084; Thermo Fischer, Waltham, MA), and incubated at 37°C in 5% CO2. Cells were seeded onto permeable polycarbonate filters (0.4-μm pore size, either 0.6 cm2 or 4.67cm2; catalog PIHP01250 and PIHP03050, respectively; Millipore Sigma, Burlington, MA) and cultured until confluent (TEER, ≥2000 Ω × 0.6 cm2 or ≥1300 Ω × 4.67 cm2). TEER was measured using an EVOM2 epithelial voltohmmeter with chopstick electrodes (World Precision Instruments, Sarasota, FL). For immunofluorescence assessment of C parvum burden, IPEC-J2 cells were seeded onto 8-well chamber slides (Nunc Lab-Tek II, catalog 154534; Thermo Fisher, Waltham, MA) and grown to confluence over a period of 3–4 days before use. IPEC-J2 cells were used at passages 38–50. Media was changed every 3–4 days.
8
Transepithelial Electrical Resistance Assay
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Isolation of primary mSCs has been described previously (Hui et al.2020 (link)). For TEER assay, primary mSCs were cultured in 6 cm dishes with 10% FBS DMEM/F-12 medium (Gibco) until monolayer was formed, then seeded cells on 12-well transwell polycarbonate membrane system (3402, Corning Life Sciences) for experiment. The cells were digested with 0.125% trypsin (Gibco) and evenly seeded at a density of 2.5 × 105 per well. The TEER was measured daily by using epithelial volt/ohm meter (Evomx) with “chopstick” electrodes (World Precision Instrument). Experiments were performed when TEER reaches 50 Ω · cm2 or more and remains unchanged, which indicates 100% cell confluency. At least 3 replicate wells were designed for each set and 3 independent experiments were repeated. The SCB permeability was expressed as the relative TEER, which represents the ratio of resistance values (Ω) as follows: (Ωexperimental condition − Ωmedium alone)/(Ωmock − Ωmedium alone).
9
Transepithelial Conductance Measurement
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Transepithelial electrical conductance was monitored dosedependently using a volt-ohm meter and chopstick electrodes, according to manufacturer's instructions (World Precision Instruments, Sarasota, FL, USA). PBS was applied for each measurement and then removed.
10
Evaluating MMP9 Effects on SCB Integrity
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As described previously [25 , 38 (link)], to evaluate the effects of MMP9 on the integrity of the SCB, 2.5 × 105 primary mSCs were plated on a 12-well Transwell polycarbonate membrane system (Corning Life Sciences, Corning, NY, USA) with a 0.4-μm pore size and a 1.12-cm2 membrane surface. The integrity of this in vitro SCB model was determined by measuring TEER using an epithelial volt/ohm meter (Evomx) with “chopstick” electrodes (World Precision Instrument) every day from day 2 after seeding. Experiments were performed with a high resistance until TEER values crossed 50 Ω • cm2, depending on the cell type, indicating 100% cell confluency [38 (link)]. Subsequently, cells were infected with ZIKV (MOI = 5) and then treated with or without the specific MMP9 inhibitor JNJ0966 for 1h. The inserts were gently washed three times with medium to remove unbound virus, and cells were treated again with the inhibitor. The TEER was measured 12, 24, 36, 48, and 72h after infection. In addition, the activated MMP9 protein with or without JNJ0966 was added to the upper chambers, and resistance values were measured at the indicated times. The SCB permeability was expressed as the relative TEER, which represents the ratio of resistance values (Ω) as follows: (Ω experimental condition –Ω medium alone) / (Ω untreated SCs –Ω medium alone).
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