To investigate the effect of GMP on E. coli translocation, Caco-2 cells were grown on transwell inserts for 21 days in DMEM containing 10% (v/v) FBS, 1% (v/v) non-essential amino acids and 0.5% penicillin–streptomycin (5000 U/mL). Prior to infection, E. coli NCTC12900 (1 × 108 CFU/mL) was pre-incubated with GMP (5 mg/mL) for 1 h at 37 °C before being applied to the apical side of the transwell plate. Serum-free media (1 mL) were added to the basolateral chamber and the cells were incubated for 14 h at 37 °C (5% CO2). Transwell inserts containing cells under the same conditions were also established for the introduction of non-treated bacteria in serum-free media (negative control). The number of translocating bacteria was determined by plating the basolateral medium onto BHI agar at 1, 2, 3, 4, 6, 8, 12 h post-apical infection as above. To confirm the formation of a tight cell monolayer, TEER measurements were carried out at 7, 14 and 21 days using an EVOM X meter coupled to an STX2 manual electrode (World Precision Instruments, Sarasota, FL, USA). To control for any disruption in the integrity of the cell monolayer during bacterial infection, TEER reads were taken at 1, 2, 3, 4, 6, 8, 12 h post-apical infection.
Evom x meter
Manufactured by World Precision Instruments
Sourced in United Kingdom
The EVOM X meter is a device designed for measuring the transepithelial electrical resistance (TEER) of cell monolayers grown on permeable membrane supports. It provides precise measurements of the electrical resistance of cell-based in vitro models, which is a crucial parameter for evaluating the integrity and barrier function of epithelial and endothelial cell layers.
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9 protocols using evom x meter
1
Investigating GMP Modulation of E. coli Translocation
2
Bacterial Infection and Barrier Integrity
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MDCK cells were plated onto transwells (5 × 104; 6.5 mm diameter; 0.4 μm—pore size; Corning) and grown until apical junctional complexes developed. Transwells were infected apically with either C. jejuni RC039, S. enterica SE 10/72 or C. perfringens ATCC® 13124™. TEER was measured at 3 h after infection using an EVOM X meter connected to an Endohm chamber (World Precision Instruments). The paracellular permeability of MDCK cells was determined using calcein as the solute as described previously [53 (link)]. Flux assay data are presented as means (SD) of triplicate independent samples performed three separate times. The presented results are representative of at least three independent experiments. The mean (SD) of at least three independent experiments for each cell line was calculated.
3
Evaluating Cryptosporidium Barrier Effects
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To investigate the effect of C. parvum G1 and G2 on the barrier properties HCT-8 cells and isolated bovine cells, transepithelial electrical resistance (TEER) was measured at 3 h postinfection using an EVOM X meter (World Precision Instruments) coupled to an Endohm chamber (World Precision Instruments), and the infected cells compared to noninfected cells whose TEER was also measured at 3 h following infection.
4
Ussing Chamber Evaluation of Intestinal Tissue
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Once the mucosal tissues were prepared, they were mounted on the vertical Ussing Chamber (Harvard Apparatus Inc., Holliston, MA, USA) as flat sheets on a 0.32 cm2 segment holder with needles for stabilisation. 5 mL KBR solution was added to each compartment of the Ussing Chamber and the solutions were gassed with an O2/CO2 (95%/5%) gas mixture. The chambers were tightly screwed and the entire assembly was controlled at 37 °C.
To evaluate tissue integrity during experiments, tissue transepithelial electrical resistance (TEER) was measured using an EVOMX meter (World Precision Instruments Inc., WPI, Hertfordshire, UK). Any jejunum tissue that showed a TEER value lower than 40 Ω·cm2 at the beginning of the experiment was regarded as poorly viable and was excluded. Whenever TEER values decreased by more than 15% from the original value (measured at the end of the 20–30 min equilibration period), the tissue was considered not to be viable and eliminated from the investigation [24 (link)].
To evaluate tissue integrity during experiments, tissue transepithelial electrical resistance (TEER) was measured using an EVOMX meter (World Precision Instruments Inc., WPI, Hertfordshire, UK). Any jejunum tissue that showed a TEER value lower than 40 Ω·cm2 at the beginning of the experiment was regarded as poorly viable and was excluded. Whenever TEER values decreased by more than 15% from the original value (measured at the end of the 20–30 min equilibration period), the tissue was considered not to be viable and eliminated from the investigation [24 (link)].
5
Measuring Epithelial Barrier Integrity
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Transepithelial resistance measures the integrity of cellular tight junctions and is a suitable method to be used in cell culture monolayers for the purpose of measuring the effect of bacteria during infection in vitro (Srinivasan et al., 2015 (link)). This methodology measures the electrical resistance, in ohms, as a measure of cellular barrier integrity. For the purpose of TEER measurement, the HCT-8 cells were grown on 0.4 μm and 12 mm pore size transwell inserts (Corning) and selected based on the formation of a confluent monolayer. Our aim was to investigate the effect of Auranta 3001 on the barrier properties of HCT-8 cells by taking TEER measurements at 3 h postinfection (±0.1 and 0.5% Auranta 3001) using an EVOM X meter connected to an Endohm chamber (World Precision Instruments).
6
Polarization of MDCK II Epithelial Cells
7
Polarization of BeWo Cells as a Placental Barrier Model
8
Measuring Epithelial Barrier Integrity Under Bacterial Challenge
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The D6234 oral primary epithelial cells were seeded in transwells (5 × 104; 6.5-mm diameter; 0.4-μm pore size; Corning) and allowed to form apical junctional complexes. Seeded and infected transwells, with either S. aureus, S. pyogenes or E. faecalis, were used to measure TEER after 3 h using an EVOM X meter connected to an Endohm chamber (World Precision Instruments). All experiments were performed at least three times. The mean (SD) was calculated for each assay.
9
Ussing Chamber Tissue Integrity Evaluation
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Once the mucosal tissues were prepared, they were mounted in the vertical Ussing Chamber (Harvard Apparatus Inc., Holliston, MA, U.S.A) as flat sheets on a 0.28 cm 2 segment holder with needles for stabilization. The mucosal surface of the tissue faced the apical chamber, while the endothelial surface faced the basolateral chamber. 5 mL blank KBR buffer was placed into each chamber, and a gas mixture of O2/CO2 (95%/5%) was bubbled through to oxygenate the liquid contents and ensure complete convection. The chambers were tightly screwed with high spring-tension retaining rings and the entire assembly was kept at 37℃with a circulating water bath for an equilibrium period of 20-30 min.
To evaluate the integrity of the mucosal tissue during experiments, tissue transepithelial electrical resistance (TEER) was measured using an EVOMX meter (World Precision Instruments Inc., WPI, Hertfordshire, United Kingdom) at half-hourly intervals. Any duodenum, jejunum, ileum and colon segments that presented a TEER value below 20Ω•cm 2 , 40Ω•cm 2 , 50Ω•cm 2 and 70Ω•cm 2 , respectively, at the beginning of the experiment was regarded as poorly viable and excluded. Whenever TEER values decreased by more than 15% from the original value (measured at the end of equilibration period), the tissue was considered not viable and eliminated from the investigation (Polentarutti et al., 1999) .
To evaluate the integrity of the mucosal tissue during experiments, tissue transepithelial electrical resistance (TEER) was measured using an EVOMX meter (World Precision Instruments Inc., WPI, Hertfordshire, United Kingdom) at half-hourly intervals. Any duodenum, jejunum, ileum and colon segments that presented a TEER value below 20Ω•cm 2 , 40Ω•cm 2 , 50Ω•cm 2 and 70Ω•cm 2 , respectively, at the beginning of the experiment was regarded as poorly viable and excluded. Whenever TEER values decreased by more than 15% from the original value (measured at the end of equilibration period), the tissue was considered not viable and eliminated from the investigation (Polentarutti et al., 1999) .
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