Dm16000b

The viability of cells attached to the microcarriers after 24 h of culture was determined by incubating the cellularized microcarriers with 10% (v/v) Presto Blue Cell Viability Reagent (Invitrogen, United Kingdom) in 2 ml proliferation medium in a 6-well plate. After incubating for 2 h at 37°C, 100 μl of the supernatant was transferred into black-walled 96-well plates (Corning, United Kingdom) and the fluorescence intensity was measured at 560 nm (exc) and 620 nm (em).
Live-Dead staining (Thermo Fisher Scientific, United Kingdom) was used to qualitatively assess cell viability on the PLGA TIPS microcarriers after 21 days incubation. Samples were imaged immediately using an inverted fluorescence microscope (Leica DM16000B).

The cells were first grown on an 8-well slide; then, they were incubated with the NucBlue® Live Cell Stain reagent (Invitrogen) for 20 min, fixed with 4% paraformaldehyde for 15 min, and incubated with 0.1% Triton 100 for 20 min. After being blocked with 2% BSA in PBS for 20 min, the cells were incubated with the following primary antibodies overnight at 4 °C: NF-κB antibodies (1:100, ab32536, Abcam), TNF-α antibodies (1:100, ab6671, Abcam), TAK1 antibodies (1:1000, ab109526, Abcam), TAB1 antibodies (1:200, ab227210, Abcam), and p-IκBα (B-9) antibodies (1:100, sc-8404, Santa Cruz Biotechnology). The cells were then washed with PBS, incubated with the corresponding secondary antibodies for 1 hour, and visualized using a fluorescent microscope (DM16000B, Leica, Heerbrugg, Switzerland). The tissues were cut into 8-μm-thick sections. After dewaxing, dehydration, and antigen retrieval, the sections were blocked using 5% serum, incubated with the primary antibodies overnight at 4 °C as mentioned above, and then incubated with biotin-labeled secondary antibodies for 1 h at room temperature.

R28 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured overnight in a CO₂ incubator. The cells were exposed to SiO₂ NPs at different concentrations (5–80 µg/mL) for 12 and 24 h. The changes in cell morphology were examined using a phase-contrast microscope (Leica DM16000B, Heidelberg, Germany).

HCEC-B4G12 cells were collected and plated in 96-well plates at a density of 1 × 10⁴ cells/well and cultured overnight in a CO₂ incubator. Next, the cells were exposed to Na-Mt, H-Na-Mt, C-H-Na-Mt, Ca-Mt, and MMt having different concentrations (1.56–100 μg/mL) for 24 and 48 h. As recently described [30 (link)], the ATP levels were measured by using the CellTiter-Lumi™ Plus Luminescent Cell Viability Assay (Beyotime, Beijing, China), while the LDH Release Assay (Beyotime, Beijing, China) was used to assess the membrane integrity of HCEC B4G12 cells, with cell viability examined by applying the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA). Luminescence and absorbance were respectively recorded in a microplate reader (SpectraMax ID3, Molecular Devices, USA). Finally, morphological changes of HCEC-B4G12 cells were also examined under a phase-contrast microscope (Leica DM16000B, Germany).

All images were acquired using a Leica TCS SP5 laser scanning microscope (Leica, DM16000 B, Wetzlar) and Leica Application Suite Advanced Fluorescence Software (LAS, AF 2.6). Multifluorescent images were obtained using the sequential scan mode. z stacks were made of each section, and crosstalk of fluorophores was eliminated automatically by the software. Images were further analyzed using ImageJ (National Institutes of Health).