Costar transwell permeable support

Primary cultures of human gingival fibroblasts (HGFs) were used. The trial was approved by the Research Ethics Committee of National Taiwan University Hospital (8 June 2011) and was registered with Case No. 201105080RC. Written informed consent was obtained from all participants collect gingival tissues. HGFs were cultured in Minimum Essential Medium alpha with 10% fetal bovine serum and 100 U/mL of antibiotics (penicillin–streptomycin–amphotericin; Sigma-Aldrich) at 37 °C in 5% CO₂. The passage number was 8–12. The specimens in each group (n = 5) were placed on the bottoms of transwell inserts (Costar Transwell Permeable Supports, Corning, NY, USA; diameter 6.5 mm, pore size 3.0 mm). For comparison of the relative toxicities of the tested materials, the transwells were transferred into 24-well culture plates, which had been seeded with HGFs at 5 × 10⁴ cells per well and allowed to adhere overnight at 37 °C. Empty inserts served as a negative control group. After incubation for 1, 3, and 5 days, the cells in each well were incubated at 37 °C for 3 h with culture medium containing 100 μL of MTT solution. The medium was then aspirated and dimethyl sulfoxide (200 μL) was added to dissolve the reduced formazan crystals. The optical density (OD₅₇₀) of the formazan solution was measured using a microplate reader (ELx 800, Biotek, Winooski, VT, USA).

Transwell migration assay was performed as previously described^{25 (link)}. Briefly, 7.5 × 10⁴ MDA-MB-231 cells or 1.0 × 10⁵ MCF-7 cells were seeded with serum-free medium into the upper chamber of 24-well Costar Transwell permeable supports with 8.0 μm pore size (Corning). The lower chamber of the well was filled with complete growth medium containing 10% FBS. Cells were cultured for 16 h (MDA-MB-231) or 48 h (MCF-7) and washed with PBS twice. Cells were fixed with 4% PFA for 15 min and washed twice with PBS. Staining was performed with 0.5% crystal violet for 15 min in the dark. Cells were washed twice with PBS and removed from the upper chamber of the insert using a cotton swab. Photos were taken with a microscope at 10 × magnification and the number of migrated cells was quantified using ImageJ.

For migration assays, EGM2 medium supplemented with 20%FBS (600 μl) was added to the lower chamber, while 5 × 10⁴ ECFCs in EGM2 medium (100 μl) were seeded on the upper chamber of Costar Transwell permeable supports (8 µm pore size, Corning, Corning, NY, USA). 4 hours after incubation at 37 °C, suspension cells were removed and the membranes were fixed with 4% paraformaldehyde for 20 minutes at RT. The migrated cells were stained with Hochest 33342 reagent (Sigma) for 5 minutes at RT and counted using fluorescence microscopy in ten random fields.
For in vitro tube formation assays, basement membrane extract (BME, Trevigen Inc., Gaithersburg, MD, USA) was coated in 96-well plates (50 μl/well) at 37 °C for 1 hour. Next, 5 × 10³ ECFCs in EGM2 medium supplemented with 20%FBS (100 μl) were placed onto the BME and incubated at 37 °C for 6 hours. 10 nM vinblastine (VB, Sigma) was added to the culture as negative control. The tube structures were visualized by inverted light microscope (100×) and ten representative fields were captured in each group. Total tube length was measured by ImageJ. Total number of tubes (>30 μm) and total number of branched tubes were counted.

Primary human bronchial epithelial cells (HBEC) from healthy donors were obtained and cultivated in an air-liquid interface system^{78 (link)}. 6 × 10⁴ cells/cm² were seeded onto collagenized (Type 1 Collagen, Merck, CC050) Costar Transwell Permeable supports (3460, Corning) in Airway Epithelial Growth Medium (AEGM, Promocell, C-21160)). After reaching confluence, they were airlifted by aspiration of apical medium. Medium in the basolateral chamber was changed to differentiation medium (DMEM (ThermoFisher, 31966021)/AEGM (1:1) supplemented with 0.1 ng/mL retinoic acid (Merck) and penicillin/streptomycin). Cells were cultivated for 28 days and barrier formation was verified by measuring transepithelial electrical resistance (TEER). All donors developed a TEER > 880 Ω/cm². Cells were used at passages 3 or 5.
Basolateral medium was exchanged for differentiation medium without antibiotics on differentiation day 26, and again immediately before infection on day 29. To assess the effect of NMN on Spn growth, Spn D39 were added apically at MOI 20 in 50 µl PBS. After one hour of incubation, cells were washed with 400 µl PBS. NMN 500 µM was added basolateral and cells were incubated for 16 h. For gene expression analysis, cells were apically infected with Spn D39 at MOI 5 or 20 in 10 µl PBS without washing for 16 h.