Transwell clear insert

MDCK cells were plated on Transwell clear inserts (pore size 0.4 μm; Corning Inc., Cat. 3460, Corning, NY, USA) and one day later were transfected in DMEM without calcium and serum with Lipofectamine™ 2000 (Life Technologies, Cat.11668-019, Carlsbad, CA, USA) with the following constructs: Flag₃-hZO-2 WT, the ubiquitination mutants FLAG₃-hZO-2-K759R and -K992R, or the SUMOylation mutant FLAG₃-hZO-2-K730R. After 6 h of incubation needed for the cells to start producing hZO-2 protein from the transfected constructs, the monolayers were switched to DMEM with 1.8 mM CaCl₂ and bovine serum. After that, the value of TER was continuously measured from each insert in the automated cell monitoring system, CellZscope (nanoAnalytics GmbH, Münster, Germany), using the CellZscope software, version 1.5.0.

OC explants were isolated from postnatal day 2 (P2) Wistar rats, P0-P2 mice (Lgr5-GFP or Atoh1-nGFP mice). Dissection of the cochlea under a stereomicroscope (Nikon SMZ800, Japan) allowed OC separation from stria vascularis and modiolus, and the sensory epithelium was plated with hair cells facing up on Cell-Tak (Corning, United States)-coated transwell-clear inserts (6-well format, 0.4 μm pore, Corning, United States). Dissection medium was exchanged for 1.5 ml full otic medium supplemented with 10% FBS (Invitrogen) and 0.01% Ampicillin (Sigma), added under the insert membrane, maintaining the OC under a thin film of medium during culture at 37°C. 5-ethyl-2′-deoxyuridine (EdU, 5 μM, Life Technologies) was added directly in the medium when indicated.

Primary airway epithelial cells (AECs) were obtained from children admitted for elective surgery for non-respiratory related conditions [21 (link)–23 ] and de-identified prior to downstream analysis. Primary AECs were then grown on 6.5-mm Transwell-Clear inserts 0.4 μm pore size (Corning, NY, USA) pre-coated with 30 μg/mL human placental collagen type I, which has been previously demonstrated to support AEC growth [24 (link)]. Cells were grown under submerged conditions in Bronchial-Air Liquid Interface (B-ALI™, Lonza, MD, USA) growth media until confluent. To differentiate into ciliated pseudostratified AECs, media was removed from the apical side and this was considered Day 0 of ALI culture and the start of the experimental period. Cells were then grown in B-ALI™ differentiation media, added to the basolateral side every alternate day and the apical side washed with tissue-culture sterile 1X PBS weekly. Cultures were grown for 28 days at ALI to ensure maximal differentiation as assessed by the presence of beating cilia as well as mucus production, as evident by mucus build-up on the apical side of the cultures.

The cell migration was measured in the Corning Transwell-Clear 24-well insert plates (Corning, Corning, NY, USA) using Polyester (PET) Membrane (8-μm pore size) Transwell-Clear Inserts (Corning, Corning, NY, USA). 3,5-DMAP-treated A549 cells (2.5×10⁴) were suspended in 0.1 mL serum-free medium, added to the upper compartment and 0.6 mL of the medium (with 10% FBS) was added to the lower compartment. After 12 h additional incubation, the A549 cells on the lower surface of PET membrane were fixed with 4% paraformaldehyde and stained with 2% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The cells were visualized with using a light microscope at 200x magnification. Migration ability of A549 cells were quantified by counting the cells that migrated to the lower side of the PET membrane with using Image J software. The migration ratio was generated by dividing the number of viable cells to the amount of control cells in the assessed region and results were presented as the fold change of the control.

NCl-H441 (American Type Culture Collection, HTB-174) cells were obtained from LGC Standards (Teddington, UK) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Fisher Scientific-UK Ltd, Loughborough, UK) at 37 °C in a humidified air atmosphere containing 5% CO₂. For establishment of an epithelial barrier, the H441 cells (1.5*10⁵ cells/insert) were cultured on the apical side of cell culture inserts in the presence of dexamethasone (Sigma-Aldrich, Poole, UK) with 1% insulin-transferrin-sodium selenite (ITS) supplement (Roche Diagnostics Limited, West Sussex, UK). The membrane supports of the cell culture inserts (0.33 cm² cell culture area) were either the PLLA polymer or PET (Transwell Clear inserts (pore size 0.4 μm), Corning, VWR, Dublin, Ireland). Responses of H441 cells to the pro-inflammatory cytokine TNFα (10ng/ml; Sigma-Aldrich, Poole, UK) were assessed on day 4 post-seeding by challenging the apical epithelial surface with the cytokine or vehicle control; after 24h the cell-free culture supernatants from the apical and basal compartments were collected and stored for ELISA, while the cells were fixed for immunofluorescence staining.

Caco-2 cells were seeded onto Transwell-Clear inserts (12-well clusters, 6.5-mm inserts with polyester membrane, pore diameter 0.4 μm, Corning NY) at a density of 10⁵ cells/insert. Each insert was placed on top of a well in a 24-well plate with 1 ml in the bottom and 200 μL media in the top as described previously (Anderson et al., 2010 (link)). Caco-2 cells were grown for 5 days until confluence in Minimum Essential Medium Eagle (MEM) with 20% fetal bovine serum (FBS) without antibiotic-antimycotic (Gibco, Carlsbad, CA, United States) at 37°C in a humidified 5% atmosphere. TEER measurements were performed using a Millicell Electrical resistance system (Millipore, Billerica, MA, United States). When monolayer of cells reached the confluence, Caco-2 cells were co-incubated with 200 μL of bacterial culture grown to OD600 0.3 (7 × 10⁷ CFU/mL) in MEM media. Consequently, the TEER was measured after 8 h of incubation.