T84 human colon cancer cells (from ATCC CCL-248) were cultured in DMEM/F12 medium (GIBCO, US) supplemented with 5% heat-inactivated fetal bovine serum (Life Technologies, US). No antibiotic supplementation was used. Cells were routinely passaged in 75 cm2 flasks (Corning, US) at 5% CO2, 37 °C. For the invasion assay, T84 cells were split into 96-well plates at a density of 105 cells/well. For polarization, T84 cells were seeded at density of 7.5 × 105 cells/well into the apical chamber of 12-well Transwells (12 mm diameter; 3 μm pore size, Corning, US). The culture medium, DMEM/F12 medium supplemented with 5% heat inactivated FBS, was replaced every 2–3 days. Transepithelial electric resistance (TER) was measured manually every 2–3 days using an epithelial voltohmmeter 2 connected to a STX2 chopstick electrode (World Precision Instruments, USA) until the reading reached above 750 Ω cm2. TER values for monolayers were calculated by subtracting the reading of an unseeded well from the seeded wells and by subsequent correction for surface area.
Stx2 chopstick electrode
Manufactured by World Precision Instruments
Sourced in United Kingdom, United States
The STX2 chopstick electrodes are lab equipment designed for microelectrode recording and stimulation. The product features a pair of fine-tipped electrodes mounted on a handle, allowing for precise positioning and manipulation during experiments. The core function of the STX2 chopstick electrodes is to facilitate the measurement and stimulation of electrical signals in biological samples or other research applications.
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Lab products found in correlation
Evom voltohmmeter, by World Precision Instruments (14 mentions)
Evom2 voltohmmeter, by World Precision Instruments (4 mentions)
Black 96 well cell culture microplate, by Greiner (4 mentions)
Evom2, by World Precision Instruments (3 mentions)
Epithelial volt ohm meter evom2, by World Precision Instruments (3 mentions)
0.4 μm pore size insert, by Greiner (3 mentions)
Glomax multi microplate multimode reader, by Promega (3 mentions)
Evom2 epithelial voltohmmeter, by World Precision Instruments (2 mentions)
75 cm2 flask, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Voltohmmeter, by World Precision Instruments (1 mentions)
Cls3460, by Corning (1 mentions)
Transwell insert, by Corning (1 mentions)
Cat no 3460, by Corning (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Collagen 4, by Merck Group (1 mentions)
Transwell filters, by Thermo Fisher Scientific (1 mentions)
37 protocols using stx2 chopstick electrode
1
Culturing and Polarizing T84 Colon Cancer Cells
2
Transepithelial Electrical Resistance Measurement
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Transepithelial electrical resistance (TEER) across the epithelium of undifferentiated and wdNHBE cells was measured using an epithelial voltohmmeter 2 (EVOM2) with an STX2 chopstick electrode (World Precision Instruments, Sarasota, FL, USA) [74 (link)]. ALI media was added to the apical and basolateral compartments, equilibrated for at least 15–20 min at RT and the TEER measured. TEER readings were membrane-corrected by subtraction of measurements from control transwells (no cells) and expressed in units of Ω or Ω × cm2.
3
Polarization of MDCK Cell Monolayers
4
Evaluating Epithelial Barrier Integrity
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Tissue integrity was assessed by measuring TEER using an EVOM2 epithelial volt-ohmmeter and an STX2 chopstick electrode (World Precision Instruments, Sarasota, FL, USA) as described previously [12 (link)].
5
Assessing Endothelial Barrier Function
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Endothelial barrier function was assessed by measurement of the TEER and TEPD. The surface of 1.12 cm2, 0.4 µm pore sized Transwell inserts (CLS3460, Corning Incorporated/Life Sciences, Tewksbury, Massachusetts, USA) were coated with FNC coating mix. Then, 1 mL of culture media was added to the bottom well of a 12-well tissue culture plate, and 200 µL was added to the inside of the collagen-coated Transwell insert. Cells were seeded onto each collagen-coated Transwell insert at a density of approximately 100,000 cells/insert and cultured at 37°C, 5% CO2 for seven days before use and transfected with siRNA. TEER and TEPD were measured using an epithelial voltohmmeter (EVOM2; World Precision Instrument, Inc., Saratoga, FL, USA). The wells were then washed twice with serum-free medium and allowed to equilibrate for one hour at 37°C, 5% CO2 before measurement.18 (link) TEER was measured in triplicate for each well. Each group contains four wells. STX2 chopstick electrode (4 mm wide and 1 mm thick; World Precision Instrument, Inc.) was used on the transwell TEER measurement.19 (link) Each experiment was repeated in triplicate.
6
Measuring Transepithelial Electrical Resistance in LEC Monolayers
7
Transepithelial Electrical Resistance Measurement
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Airway epithelium integrity was measured by transepithelial electrical resistance (TEER) before (day 0) and 24 h after each exposure. First, 200 µL of MucilAirTM culture medium was added to the apical surface of each insert, and then the measurements were carried out with an EVOM2 voltohmmeter with an STX2/chopstick electrode (World Precision Instruments, Sarasota, FL, USA), in agreement with manufacturer’s recommendations. The resistance value appeared on the EVOMX screen and TEER (Ω·cm2) was then calculated using the following Formulas (1) and (2) [35 (link)].
where R is resistance, Ω; Rmembrane is membrane resistance = 100 Ω; and A is the surface area of the porous membrane of the insert = 0.33 cm2. According to Epithelix Sàrl, TEER values were typically in the range of 200–600 Ω·cm2. Afterward, the culture medium was gently aspirated from the apical surface and MucilAirTM–HF inserts were immediately exposed to STW, ISCS, or clean air. Inserts that were not exposed were returned to the incubator.
where R is resistance, Ω; Rmembrane is membrane resistance = 100 Ω; and A is the surface area of the porous membrane of the insert = 0.33 cm2. According to Epithelix Sàrl, TEER values were typically in the range of 200–600 Ω·cm2. Afterward, the culture medium was gently aspirated from the apical surface and MucilAirTM–HF inserts were immediately exposed to STW, ISCS, or clean air. Inserts that were not exposed were returned to the incubator.
8
16HBE Epithelial Barrier Function Assay
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16HBE cells were seeded at a density of 105 cells per well on permeable transwell inserts with a polyester membrane with a diameter of 12 mm and a pore size of 0.4 µm (cat. no. 3460, Corning, Amsterdam, Noord-Holland, The Netherlands). Before seeding, the apical side of the insert was coated with a 30 µg/mL bovine collagen type I solution in 70% ethanol. The solution was filtered, after which the insert was coated by applying 70 µL to the apical side of the transwell. Then, the ethanol was allowed to evaporate overnight in a laminar flow hood under exposure to UV light to preserve sterility. The 16HBE cells were grown under liquid-covered culture conditions wherein the apical and basolateral fluid volumes were set at 250 and 900 µL, respectively. The epithelial barrier function of 16HBE was evaluated by measuring the trans-epithelial electrical resistance (TEER) using an EVOM volt–ohmmeter and an STX2 “chopstick” electrode (World Precision Instruments, Friedberg, Germany). The TEER values measured were corrected for the background resistance of an empty insert containing only medium and calculated as Ω·cm2.
9
Evaluating Tissue Barrier Integrity by TEER
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The effect of FA on tissue barrier integrity was evaluated at T5 and PT10 by measuring changes in TEER using an EVOM2 epithelial volt-ohmmeter fitted with a STX2 chopstick electrode (World Precision Instruments, Sarasota, FL). Prior to the measurement, cultures were equilibrated to room temperature and the EVOM2 was calibrated to 1000 Ω using a test electrode. DPBS was added to both the apical (200 µL) and basolateral sides (400 µL). Three measurements spaced at 120° from each other were made for each culture and the average was calculated for data analysis.
10
Transepithelial Electrical Resistance Measurement
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TEER was evaluated using the EVOM2 voltohmmeter with STX2 chopstick electrodes (World Precision Instruments). TEER was measured 1 day after iBMECs were subcultured onto cell culture inserts (ThinCerts) and approximately every 24 h thereafter. A ThinCert coated with the ECM proteins, but without cells, was used to subtract the medium and membrane effects on TEER. The reported values are multiplication of surface area and measured electrical resistance of the iBMEC layer to eliminate the effect of cell culture area on the electrical resistance.
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