Costar transwell

Primary airway epithelia (passage 0) were isolated from human donor bronchi and grown at air-liquid interface on collagen-coated, semi-permeable membranes with a 0.4 μm pore size (Costar Transwell; surface area 0.33 cm²; Corning) as reported previously (15 (link)). Human airway epithelial cultures were grown in DMEM/F12 with 2% Ultroser G (USG) media at 37°C with 5% CO₂. All cell preparations used in this study were well-differentiated (>3 weeks old; resistance >1,000 Ohm x cm²). Using this culture method, we find that each Transwell filter typically supports approximately 300,000 cells. The study was approved by the Institutional Review Board at the University of Iowa. Calu-3 cells were first cultured in MEM supplemented with 20% FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 2 mM l-glutamine, 1% penicillin and streptomycin, and 0.15% NaHCO₃ at 37° C with 5% CO_2. When cells reached 80% confluence in submerged culture conditions, the Calu-3 cells were transferred to collagen-coated Transwell membranes as described (15 (link)) to generate air-liquid interface (ALI) cultures. Vero 81 cells were maintained in DMEM with 10% FBS and 1% penicillin and streptomycin at 37°C with 5% CO_2.

Chemotaxis assays were performed in a modified Boyden’s chamber with 8-μm pore polycarbonate membrane inserts (Costar Transwell; Corning Costar, Lowell, MA, USA) as described previously. In brief, cells detached with 0.25% trypsin were seeded into the upper chamber of an insert at a density of 6 × 10⁴ in 100 μl. The lower chamber was filled with pre-warmed culture medium containing test reagents. Medium supplemented with 0.5% BSA was used as a negative control. After 24 hours, the inserts were removed from the Transwell supports. The cells that had not migrated were scraped off with cotton swap from the upper membrane, and the cells that had transmigrated to the lower side of the membrane were fixed and stained with HEMA 3 (protocol, Fisher Scientific, Pittsburgh, PA) and counted on the lower side of the membrane using an inverted microscope.

Hek293LTV, CaCo2 WT (ATCC, HTP-37), and KI cells were cultured in DMEM (Sigma-Aldrich) containing high glucose, sodium pyruvate, 100 U/ml penicillin (Sigma-Aldrich), 100 µg/ml streptomycin (Sigma-Aldrich), 5% nonessential amino acids (Gibco), and 10% FBS (Gibco) in a humidified atmosphere with 5% CO2 at 37°C. For experiments requiring fully polarized growth conditions, CaCo2 cells were seeded on 24 mm or 75 mm filters (Costar Transwell; pore size of 0.4 µm; Corning) and cultured for 14–28 days. For 3D cyst assays, CaCo2 cells were cultivated and processed as described previously (Vogel et al., 2015b (link); Jaffe et al., 2008 (link)). For this purpose, 5 × 10⁴/mL single cells were embeeded in Matrigel (BD Biosiences, #356231), plated on 8× chamber slides (Lab-Tek-chamber slide, Sigma) chamber slides (10,000 cells/slide) and grown for 7 days (Román-Fernández et al., 2018 (link)). All cell lines were regularly tested negative for mycoplasm.