MDCK cells (CCL-34 and _frt transfectants) were grown on 12 mm Millicell-HA filters with a pore size of 0.45 μm (Millipore, Billerica, MA). 105 cells were seeded on each filter and subsequently cultured in 24-well trays for 2–4 days to obtain a confluent monolayer. Tightness of the cell layer was evaluated each day with a modified fluorescein leakage test [46 (link)] where 10 μg/L sodium-fluorescein (Sigma-Aldrich) was added to the apical side of cells for 15 min. at 37°C before measuring the degree of leakage into the basolateral media fraction using an ELISA reader. The cell layer was considered tight when the leakage was less than 5% compared to a control filter without cells. Cells grown on 0.4 μm PET 12 well hanging culture inserts (Millipore) were evaluated by trans-epithelial electrical resistance measurements. Briefly, filters were placed in an Endohm-12 culture cup (World Precision Instruments, Sarasota, FL), and resistance was measured in triplicates according to manufacturers instructions using an EVOM voltohmmeter (World Precision Instruments). Cells with background-substracted resistances above 100 Ω were considered tight.
Endohm 12 culture cup
Manufactured by World Precision Instruments
Sourced in United States
The Endohm-12 culture cup is a laboratory equipment product designed for culturing cells. It provides a controlled environment for cell growth and experimentation. The Endohm-12 features a 12-well configuration and is made of materials suitable for cell culture applications.
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2 protocols using endohm 12 culture cup
1
Evaluating MDCK Cell Monolayer Tightness
2
Evaluating Caco-2 Cell Monolayer Integrity
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The permeability of lucifer yellow at a concentration of 100 μM was utilised as a passive permeability marker. Lucifer yellow Papp was below 1.1 × 10−6 cm/s and was not affected by increasing concentrations of zosuquidar.
Before and after each experiment, transepithelial electrical resistance (TEER) was measured to assess cell monolayer integrity with an EVOM2 epithelial voltohmmeter connected to an Endohm 12 culture cup (World Precision Instruments, Sarasota, FL, USA). The TEER of Caco-2 cells before the experiments ranged from 557 to 747 Ω × cm2 and dropped by 42% on average after the experiment. No single filter displayed a drop in TEER of more than 65% during the experiment. TEER measurements were consistent with previous in-house studies (Al-Ali et al., 2018a (link)). Based on the TEER measurements and lucifer yellow Papp, all cell layers were considered intact, and treatments were not considered to affect cell layer integrity.
Before and after each experiment, transepithelial electrical resistance (TEER) was measured to assess cell monolayer integrity with an EVOM2 epithelial voltohmmeter connected to an Endohm 12 culture cup (World Precision Instruments, Sarasota, FL, USA). The TEER of Caco-2 cells before the experiments ranged from 557 to 747 Ω × cm2 and dropped by 42% on average after the experiment. No single filter displayed a drop in TEER of more than 65% during the experiment. TEER measurements were consistent with previous in-house studies (Al-Ali et al., 2018a (link)). Based on the TEER measurements and lucifer yellow Papp, all cell layers were considered intact, and treatments were not considered to affect cell layer integrity.
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