A549 cells (ATCC) from passages 5–15 were cultured in RPMI 1640 (Gibco) medium supplemented with 10% FBS (Atlanta Biologicals, lot C0089) and 1% penicillinstreptomycin, then incubated at 37°C in a humidified 5% CO2 environment. Upon reaching confluence (5–7 days), cells were rinsed with phosphate-buffered saline (PBS) and treated with a 0.25% Trypsin-EDTA solution (Gibco) for 8 min to dissociate cells. Cells were suspended in culture medium and 0.5 mL aliquots were seeded onto 12 mm transwell inserts (Corning, 0.4 μm pore size, polycarbonate) at a concentration of 1.25×105 cells/mL with 0.53 mL of modified RPMI 1640 under the basal side of each transwell. The cells were grown to confluency under submerged conditions for 24 h. The apical media was then aspirated, and cells were allowed to adapt to an air interface for 12 h according to previously established methods (Kim et al., 2013 (link); Jing et al., 2015 (link)).
12 mm transwell inserts
Manufactured by Corning
Sourced in United States
The 12 mm Transwell inserts are a laboratory product designed for cell culture applications. They feature a semipermeable membrane that allows the passage of nutrients, signaling molecules, and other substances between the upper and lower chambers of the insert. The inserts provide a controlled environment for studying cell migration, invasion, and other cellular processes.
Automatically generated - may contain errors
4 protocols using 12 mm transwell inserts
1
Air-Liquid Interface Culture of A549 Cells
2
C. acnes Impacts Keratinocyte Barrier Function
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Confluent HPV-KER monolayer cultures were grown on 12 mm transwell inserts (pore size 0.4 µm, Corning, New York, USA) in KSFM media. Confluent Ca-low cultures were treated with C. acnes 889 strain (MOI = 300) and transepithelial electrical resistance (TEER) values were measured in Hank’s salt balanced solution (HBSS) using an Epithelial Volt/Ohm (TEER) Meter EVOM2 (World Precision Instruments, Sarasota, FL, USA) at 24 and 72 h after treatment.
For the Ca-high cultures, confluent, contact-inhibited keratinocyte cultures were further differentiated by the addition of Ca2+ (1,7 mM) to the KSFM culturing media and incubation for 72 h. TEER values were measured at 24 and 72 h after treatment with C. acnes 889 in HBSS buffer using an Epithelial Volt/Ohm (TEER) Meter EVOM2 (Sarasota, FL, USA).
For the Ca-high cultures, confluent, contact-inhibited keratinocyte cultures were further differentiated by the addition of Ca2+ (1,7 mM) to the KSFM culturing media and incubation for 72 h. TEER values were measured at 24 and 72 h after treatment with C. acnes 889 in HBSS buffer using an Epithelial Volt/Ohm (TEER) Meter EVOM2 (Sarasota, FL, USA).
3
Co-culture of BV-2 and HT22 Cells
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BV-2 and HT22 cells were co-cultured in 12 mm transwell inserts with 0.4 μm pore size (Corning). BV-2 cells were maintained on the transwell insert and HT22 cells were kept in a 24-well plate for 24 h of co-culture before further experiments.
4
Evaluation of e-Liquid Nicotine Exposure
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NHBE cells were isolated from appropriately consented donors whose lungs were found unsuitable for transplantation. The 15 donors were non-smokers with no known airway diseases, 5 females and 10 males aged 25–58 years. From a racial aspect, there were 10 Caucasians, 2 African-Americans, 1 Hispanic white and 2 unknowns. After expansion, cells were redifferentiated at the air–liquid interface (ALI) on collagen-coated 12-mm Transwell inserts (Corning, Lowell, Massachusetts, USA).17–19 (link) The cells were exposed to 36 puffs of vaporised e-liquid either containing 0 or 36 mg/mL nicotine using the VC-1 exposure system (Vitrocell Systems, Waldkirch, Germany), with a 70 mL puff volume applied every 30 s. Cells were also exposed to nicotine (0 or 100 μM, with an estimated final 100 nM nicotine concentration in the airway surface liquid (ASL) after nebulisation) aerosolised in the Cloud system (Vitrocell Systems) or baseolaterally supplemented nicotine (0, 1 or 10 μM) for 12 hours or 5 days.
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