Evaluating Cytotoxicity of Clay Nanoparticles

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).