Matrigel growth factor reduced gfr basement membrane matrix

The human airway organoid culture method was slightly modified from a previous report (Sachs et al, 2019). In brief, human bronchial/tracheal epithelial cells (502‐05a, Cell APPLICATIONS) were suspended in 10 mg/ml cold Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix (356230, CORNING), and 50 μl drops of the cell suspension were solidified on prewarmed 24‐well culture plates at 37°C with 5% CO₂ for 10–20 min. Five hundred microliters of airway organoid medium (composition is shown in Reagent Table) was added to each well, and the medium was changed every 2–3 days. Airway organoids were passaged every 2 weeks. For passaging, airway organoids were first mechanically sheared through a flamed glass Pasteur pipette and further dissociated by incubation with TrypLE select enzyme (12563011, Thermo Fisher Scientific). The dissociated organoid fragments were collected by centrifugation at 400 g for 5 min and reseeded as above at the ratios of 1:2 to 1:4.

Caco-2 cells (Passage #47) and FITC-dextran were purchased from Millipore Sigma (Sigma-Aldrich Inc, St. Louis, MO, USA). Propranolol hydrochloride, metoprolol tartrate, and atenolol were obtained from Tocris Bioscience (Bio-Techne Corporation, Minneapolis, MN, USA). Corning^® Transwell™ 24-well plates with permeable polyester membrane inserts (6.5 mm diameter and 0.4 μm pore size), GIBCO™ TrypLE™ express enzyme (1X, no phenol red), trypan blue solution (0.4%), Corning™ Matrigel^® growth factor reduced (GFR) basement membrane matrix (phenol-red-free, LDEV-free) for organoid culture, Fetal bovine serum (FBS), and GIBCO Hank’s balanced salt solution (HBSS) were purchased from Thermo Fisher Scientific (Bedford, MA, USA).

Ham’s F12 nutrient mix, Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), penicillin/streptomycin, and Lab-Tek II 8-well chamber slide were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Corning^® Matrigel^® Growth Factor Reduced (GFR) basement membrane matrix and cell recovery solution were acquired from Corning (Corning, NY, USA). Ribonuclease A and phenylmethylsulphonyl fluoride (PMSF) protease inhibitor were purchased from Sigma Chemicals (St. Louis, MO, USA). Bradford solution was purchased from Bio-Rad (Hercules, CA, USA). Hoechst 33342, TRITC-conjugated phalloidin, primary antibodies against galectin-3, ZEB-1, N-cadherin, E-cadherin, β-catenin, vimentin, horseradish peroxidase (HRP)-conjugated secondary antibodies against anti-rabbit and anti-mouse antibodies, Alexa Fluor^® 647-conjugated secondary antibody against anti-rabbit antibodies and SignalFire™ ECL Reagent were purchased from Cell Signaling Technologies (Denver, MA, USA). The primary antibody against β-actin was purchased from Sigma-Aldrich. Triton X-100 was obtained from Bio-Rad. Recombinant human galectin-3 (rGal-3) was purchased from Merck (Dorset, UK).

Invasion assay was performed using permeable cell culture inserts (Thomas Scientific, Swedesboro, NJ) coated with 30 µl of Corning^® Matrigel^® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, NY). Inserts were incubated for 30 min at 37 °C to allow the matrigel to solidify. We followed a procedure similar to the migration assay described above but utilized matrigel coated inserts.

The small intestine was flushed with ice-cold phosphate buffered saline and cut into 1 cm segments and then transferred into BSS buffer (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mM KH₂PO4, 5.6 mM Na₂HPO4 and 15 mM EDTA) (isolation buffer), vortexed at 4 °C for 10 minutes, transferred into fresh isolation buffer, and vortexed for another 10 minutes. The crypts and villi were separated via a 70 µm cell strainer, spun down at 500 g for 3 minutes and washed once with ice-cold PBS. For ex vivo stimulation, the enterocytes were resuspended in DMEM/F12 medium and stimulated with 50 ng/ml IL-4 (#5208, Cell Signaling Technology, Danvers MA) or IL-13 (#210-13, PEPROTECH, Rocky Hill NJ) in a tissue culture incubator for 10 minutes. The enterocytes were collected, lysed with sodium dodecyl sulfate sample buffer and analyzed by Western blot assay. For organoid culture, the crypt fraction was mixed with matrigel (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, #356231, Corning NY) and the standard protocol^{17 (link)} was followed to generate organoids using IntestiCult™ Organoid Growth Medium (Mouse) (#06005, STEMCELL Technology, Cambridge MA). To induce tuft cell differentiation, the organoids were stimulated with 50 ng/ml IL-4 for at least 48 hours. The organoids were then fixed for immunofluorescence, or harvested for protein & mRNA analysis.