Eclipse ts2 fl

A field emission-scanning electron microscope (FE-SEM, JSM-7500F JEOL), high-resolution transmission electron microscope (TEM, SU900; operating at a band gap of 0.8 eV and acceleration voltage of 300 kV), and optical microscope (Eclipse Ts2-FL, Nikon) were used to determine the surface morphology and characteristics of the HINS samples. The dynamic light scattering technique was used to measure the zeta potential of the material and its modified equivalent on the DynaPro NanoStar instrument (Wyatt). Light microscopy was performed by the Leica Microsystems (Ltd., Milton Keynes, UK; magnification, ×100 and ×200). Then, the basic characterization studies (UV-Visible, XRD, FTIR, and EDX) of HINS composite has been performed in the previous study.²⁶ (link)

Cell invasion assays were performed using the QCM ECMatrix Cell Invasion Assay kit (ECM550) (EMD Millipore Corp., Billerica, MA, USA) according to the manufacturer’s instructions. Briefly, RPMI 1640 containing 20% FBS was added to the lower chamber of a 24-well plate. An invasion chamber containing an 8-µm polycarbonate membrane layered with ECMatrix^TM was aseptically inserted into the 24-well plate. AGS cells were prepared in serum-free media containing OTEO (IC₅₀), then 300 µL of the cell suspension (1 × 10⁶ cells/mL) was subsequently added into the invasion chamber. Serum-free medium without OTEO was used as control. The 24-well plate was incubated at 37 °C for 24 h in a CO₂ incubator, after which non-invading cells were removed from the invasion chamber and the invasive cells were stained with crystal violet for 20 min. Stained invasive cells at the lower surface of the invasion chamber were observed and counted under an inverted microscope (ECLIPSE Ts2-FL, Nikon, Tokyo, Japan).

Intracellular calcium was assessed with a Fluo-4 AM dye (Abcam; Cambridge, United Kingdom) and confocal microscopy, following the manufacturer’s instructions. Fluo-4 AM was diluted in DMSO containing 2 mM probenecid and 0.02% pluronic F-127. In brief, the SH-SY5Y cells (2.2 × 10⁴ cells/mL) were cultured in black, 96-well plates for quantification and in 6-well plates for imaging in DMEM/F12 without phenol red. Then, 60–70% confluence cells were stimulated with H₂O₂ (200 µM) for 2 h 30 min in serum-free media. The cells were washed with PBS containing probenecid (2 mM) at room temperature. The cells were incubated with the Fluo-4 AM (2 µM) dye loading solution at 37 °C for 30 min and protected from light, then washed with PBS containing probenecid (2 mM) at room temperature for 30 min. For the 96-well plates, the fluorescence intensities (λ_ex = 488/λ_em = 515 nm) were measured using a fluorescence microplate reader (Molecular Device; Sunnyvale, CA, USA), and confocal images were acquired with a Nikon Eclipse Ts2-FL diascopic and epi-fluorescence illumination microscope.

All barrier formation experiments were performed using a liquid-liquid interface culture system (Figure 2) in a transwell chamber, partitioned into apical and basolateral compartments by an inserted basket (insert). In this experiment, 16HBE cells (1 × 10⁵ cells/well) were seeded onto the upper surface of an insert-well membrane (diameter 6.5 mm, pore size 0.4 μm) in 24-well transwell plates. The cells were cultured at 37°C under 5% CO₂ for 7 days in the normal medium, which was added to both the apical and basolateral compartments. The medium was replaced every day. Cell morphology or confluency was observed daily using a stereomicroscope (ECLIPSE Ts2-FL; Nikon, Tokyo, Japan). TEER values and Na-F permeability were measured daily.

The cell migration assay was performed using the CytoSelect™ Wound Healing Insert (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the plastic insert was added into a well of a 24-well plate, then the cells were seeded at a density of 1 × 10⁶ cells/well. After incubating for 12 h, the insert was removed carefully to create a 500 μm-width cell gap for measuring migration rates of the cells. Serial dosages of IGF-1 ranging from 0.325 to 2.6 pmol in the hyperglycemic medium was tested to determine the optimal dosage of IGF-1 in vitro. The SF-films loaded with different amounts of IGF-1 were placed directly on top of the cell monolayer with a created gap in medium containing either 25 mM (regular medium) or 50 mM (hyperglycemic medium) of glucose. Wound closure was serially imaged at different times (0, 3, 6, 8, 10, and 13 days) post-scratching using an inverted light microscope (ECLIPSE Ts2-FL, Nikon, Tokyo, Japan). Autoclaved alamarBlue reagent-containing medium was used as a positive control according to the kit manufacturer’s instruction. The fluorescence of the culture supernatant, which reflected the cell metabolic activity, was determined with excitation and emission wavelengths of 570 and 600 nm, respectively. The cell proliferation rate was calculated according to the formula provided by the dye manufacturer.