Inverted optical microscope

HUVECs were purchased from American Type Culture Collection (ATCC, USA), and 2 × 10³/well cells were seeded in 96-well plates. After cultured with 100 µl FBS-free medium for 12 h, cells were then treated with ADSC-CMs from different group for 24 h, 48 h and 72 h. CCk8 assay was performed to detect the relative cell viability. And migration capacities of HUVECs cultured in different ADSC-CMs were evaluated using a Trans well assay. The detailed protocols were the same as those for ADSCs described above.
A wound healing assay was used to assess the migration of HUVECs as well. Scratches were created using 200-µl sterile pipette tips in six-well culture plates when the cells had grown to 100% confluence. After scratching, the cells were washed with PBS and cultured in ADSC-CMs. Photographs were taken 0 h and 24 h post-culture using an optical inverted microscope (Nikon, Japan).

The migration and invasion abilities of gastric cancer cells were assessed using Transwell assays (Millipore, Billerica, MA, USA). GCs were seeded into uncoated or Matrigel-coated (BD Bioscience, Bedford, MA, USA) plates with 8-μm diameter pores in order to perform migration and invasion assays, respectively. The top chambers were seeded with cells at a density of 2 × 10⁴ cells/well in serum-free medium, while FBS with 10% serum was added to the lower chamber. After 24 h of incubation, nonmigratory cells found on the top surface of the filter were removed by rubbing with a cotton swab. Cells that had migrated to the lower chamber were quantified in five random fields using an optical inverted microscope at ×200 magnification (Nikon, Tokyo, Japan).

For cell migration, 2 × 10⁵ CRC cells with gradient concentrations of RP4 at concentrations of 0 μM, 10 μM, 20 μM, and 40 μM were seeded into each Transwell chamber, and the underlayer was supplemented with complementary medium supplemented with 20% FBS. For the invasion assay, Transwell chambers were coated with Matrigel (Corning, New York, NY, USA) in advance to mimic the cellular matrix. The following steps were the same as those used in the cell migration assay. Migrating and invasive cells were visualized using 0.5% crystal violet and captured using an optical inverted microscope (Nikon, Tokyo, Japan).

Alizarin Red S (Sigma-Aldrich) was used in order to identify cultures’ mineralization 14 days after the induction of osteogenesis. At the end of the culturing time, cells were washed twice in PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at RT, thus rinsed twice in ddH₂O and covered with a solution of Alizarin Red S 40 mM (pH 4.2) prepared in ddH₂O. Cultures were subsequently incubated for 40 min at 4 °C on an orbital shaker. After extensive washing with ddH₂O, specimen were air-dried before observation with an optical inverted microscope (Nikon).
After images acquisition Alizarin Red S concentration was measured by its solubilization. To this purpose, each well was treated with 400 μL of 10% acetic acid (Sigma-Aldrich) and incubated for 30 min at RT under shaking. Cells were then scraped from the plate, vortexed vigorously for 30 s, heated to 85 °C for 10 min, transferred on ice for 5 min and centrifuged at 20,000 rpm for 15 min at RT. After centrifugation, the supernatants were transferred into new eppendorf tubes, and the solution neutralized by adding 75 μL of 10% ammonium hydroxide. Sample absorbance was measured at 405 nm with a Multiskan^® FC microplate reader (Thermo Fisher Scientific, Carlsbad, CA, USA) and compared to a standard Alizarin Red S curve.

The circular dichroism spectra were obtained by measuring the optical transmission of right- and left-handed chiral metasurfaces, under right- and left-handed circularly polarized illumination, in a Nikon inverted optical microscope, equipped with a halogen lamp (as shown in Fig. 2b). The polarizations of the incident light were prepared by sending the light through a polarizer and a broadband λ/4 waveplate (Thorlabs AQWP05M-580) with nearly achromatic transmission, 0.95 < T < 0.98, and retardance, 0.24 < ρ < 0.26, in the spectral region investigated. The light was focused on the sample using a Nikon LWD Achromat Condenser, with 10 mm working distance and adjustable NA. To ensure light collection from a spot smaller than the metasurface array, we used a Nikon ×100 objective with 0.7 NA and a multimode optical fiber, acting as pinhole. The spectra were recorded using an Acton SpectraPro 2300i monochromator and spectrograph.