TEER was measured using a EVOM voltohmeter connected to an Endohm 9 electrode chamber (World Precision Instruments, Sarasota, FL) as previously described [29 (link)]. Tissue culture inserts containing monolayers of HGEc cultured on Transwell-Collagen-coated membrane, were placed in the chamber and the TEER was recorded after 10 sec [29 (link)]. The permeability experiments were done as described before [31 (link)]. Briefly, 3 x 105 HGEC-1 were plated onto Transwell Collagen-coated membrane inserts (Corning Costar, Cat No 3415) in DMEM media with 10% FBS, and left for 3 days to form mature monolayer with a trans-endothelial electrical resistance (TEER) of ~ 60 ± 10 Ω /cm2. Subsequently, the cells were starved for 5 hours in serum free DMEM media without phenol red, treated with the corresponding reagents, and incubated with 1 mg/ml FITC-dextran (Molecular Probes, D-1844, Invitrogen) for 30 min at 37°C. Samples collected from the bottom chambers were read in triplicates on the Victor 3V1420 multi-counter (Perkin-Elmer, Wellesley, PA).
Evom voltohmeter
Manufactured by World Precision Instruments
Sourced in United Kingdom
The EVOM voltohmeter is a device used to measure the electrical properties of cell cultures and other biological samples. It is designed to provide accurate and reliable measurements of transepithelial electrical resistance (TEER) and short-circuit current (Isc). The EVOM voltohmeter is a versatile tool that can be used in various research and laboratory settings.
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4 protocols using evom voltohmeter
1
Measuring Endothelial Cell Permeability
2
Epithelial Permeability Measurement
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Ionic permeability was measured as transepithelial electrical resistance (TER) using chopstick electrodes with an EVOM voltohmeter (World Precision Instruments, Aston, UK). Data are expressed as ohms.cm2 and have been corrected for the resistance of an empty Transwell®. Macromolecular permeability was measured 3 and 21 hours after dsRNA challenge by adding FITC-labeled dextran to the apical compartment of co-cultures; FITC-dextran flux into the basolateral compartment was quantified 3h later by spectrofluorometry.
3
Transwell Membrane Characterization Protocol
4
Assessing Brain Barrier Integrity
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Transepithelial/transendothelial electrical resistance (TEER) were used to assess the brain integrity using an EVOM Volt-Ohmeter (World Precision Instruments). STX2 probes were disinfected and air-dried before being inserted into upper and bottom chambers. Resistance was measured for empty wells containing media before determined across monolayers. Values were calculated by multiplying raw values by transwell growth area once the base value for empty wells had been subtracted. 70,000 cells per 24-well transwel insert were seed and allow them to grow for 4 days in control and PRELP conditioned media. TEER measurements were completed 96 h after PRELP treatment.
For the membrane permeability, permeability assays were completed using an in vitro vascular permeability assay kit (Merck). In brief, inserts were hydrated and seeded with 0.5 × 105 cells/insert in media and incubated until a monolayer formed. PRELP conditioned media was applied and incubated for 24 h. FITC-Dextran was added to each insert and incubated for 20 min in the dark before the insert was removed from the well. Media in the well was mixed and transferred to black 96-well opaque plate to measure fluorescence intensity at 485 nm and 535 nm excitation and emission respectively.
For the membrane permeability, permeability assays were completed using an in vitro vascular permeability assay kit (Merck). In brief, inserts were hydrated and seeded with 0.5 × 105 cells/insert in media and incubated until a monolayer formed. PRELP conditioned media was applied and incubated for 24 h. FITC-Dextran was added to each insert and incubated for 20 min in the dark before the insert was removed from the well. Media in the well was mixed and transferred to black 96-well opaque plate to measure fluorescence intensity at 485 nm and 535 nm excitation and emission respectively.
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