Use of Oxodish sensor plates for real-time in vitro oxygen consumption has been previously described (Kelly et al., 2015 (link)). Briefly, Caco-2 cells were grown to confluence on Transwell semi-permeable inserts (0.4μm, Corning# 3470). Cells were then washed and placed into Hanks’ Balanced Salt Solution containing Ca2+/Mg2+ and supplemented with 10mM HEPES (HBSS+) (Cartwright et al., 2021 (link)). Cells were then incubated on an Oxodish sensor plate with or without apical STm, and the percent O2 of the surrounding buffer was measured over time. Note that due to the high altitude (>5000 ft.) of the laboratory where experiments were performed, fully oxygenated air is less than the ~21% observed at sea level. Control wells included HBSS+ only, as well as wells containing HBSS+ + bacteria without IECs.
Transwell semi permeable inserts
Manufactured by Corning
Transwell semi-permeable inserts are laboratory equipment used to study the movement of cells, molecules, or substances across a barrier. These inserts consist of a permeable membrane that is suspended within a well, allowing for the separate compartmentalization of different solutions or cell cultures.
Automatically generated - may contain errors
4 protocols using transwell semi permeable inserts
1
Real-time Oxygen Consumption in Caco-2 Cells
2
Differentiation of Human Airway Epithelial Cells
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Human bronchial and tracheal epithelial cells were obtained from residual tissue from lungs destined for transplantation in collaboration with the University of Wisconsin - Health Lung Transplant Program. The protocol was reviewed by the University of Wisconsin Institutional Review Board and was deemed “not human subjects research.” Cryopreserved aliquots of cells were thawed and expanded as monolayers in PneumaCult-Ex Plus Medium (StemCell Technologies) supplemented with 0.1% [vol/vol] gentamicin (Sigma) and 0.1% [vol/vol] fluconazole (Novaplus). Once the cells reached 80% confluence, they were transferred to 12-well plates with Transwell semi-permeable inserts (Corning 3460) and were allowed to differentiate in PneumaCult-ALI medium (StemCell Technologies) supplemented with 0.1% [vol/vol] gentamicin (Sigma) and 0.1% [vol/vol] fluconazole (Novaplus) at the air-liquid interface for at least 21 days when ciliary motion was observed. The culture medium was changed to an antibiotic-free medium containing 0.05% [vol/vol] hydrocortisone 48 hours prior to RV infection. All ALI cultures were used between 28 – 35 days of air lifting.
3
Real-time Oxygen Consumption in Caco-2 Cells
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Use of Oxodish sensor plates for real-time in vitro oxygen consumption has been previously described (Kelly et al., 2015 (link)). Briefly, Caco-2 cells were grown to confluence on Transwell semi-permeable inserts (0.4μm, Corning# 3470). Cells were then washed and placed into Hanks’ Balanced Salt Solution containing Ca2+/Mg2+ and supplemented with 10mM HEPES (HBSS+) (Cartwright et al., 2021 (link)). Cells were then incubated on an Oxodish sensor plate with or without apical STm, and the percent O2 of the surrounding buffer was measured over time. Note that due to the high altitude (>5000 ft.) of the laboratory where experiments were performed, fully oxygenated air is less than the ~21% observed at sea level. Control wells included HBSS+ only, as well as wells containing HBSS+ + bacteria without IECs.
4
Culturing Human Airway Epithelial Cells
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Human bronchial and tracheal epithelial cells were obtained from residual tissue from lungs destined for transplantation in collaboration with the UW Health Lung Transplant Program. The protocol was reviewed by the University of Wisconsin IRB and was deemed “not human subjects research.” We thawed cryopreserved aliquots of cells and expanded them as monolayers in PneumaCult-Ex Plus Medium (StemCell Technologies) supplemented with 50 μg/mL gentamicin and 2 μg/mL fluconazole at 37°C in a 5% CO2 atmosphere. Once the cells reached 80% confluence, we transferred them to 12-well plates with Transwell semipermeable inserts (Corning) and allowed them to differentiate in PneumaCult-ALI medium (StemCell Technologies) supplemented with gentamicin and fluconazole, as above, at the ALI until ciliary motion was observed (≥21 days). We cultured the cells in an antibiotic-free medium 48 h prior to bacterial inoculation.
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