Bisbenzimide h33342 fluorochrome trihydrochloride

Cells cultured in 24-well plates were exposed to 10 μg/mL CNMs for 24 h. After washing the cells three times with DPBS, cells were stained with bisbenzimide H33342 fluorochrome trihydrochloride (H33342, 10 µg/mL; Nacalai Tesque, Kyoto, Japan) and CytoPainter Lysosomal Staining Kit Orange Fluorescence (CytoPainter ab 138895; Abcam, Cambridge, UK) for 1 h. The cells were visualized using an AxioObserverZ1 fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40 × objective lens.

The pattern of cell attachment on the surface of CNTp scaffolds were observed by fluorescence microscopy and SEM.
MC3T3-E1 preosteoblasts were trypsinized using a 0.25% trypsin/EDTA solution (Sigma, Saint Louis, MO, USA), seeded onto scaffolds in 96-well plates at a density of 1 × 10⁴ cells/scaffold, and suspended in 20 μL of media. They were incubated in a CO₂ incubator for an hour at 37 °C and 5% CO₂ saturated humidity until cells adhered to the scaffold. Each scaffold was then transferred to other 96-well plates and cultured by adding 200 μL of alpha-MEM. Media were replaced twice a week.
On day 5, scaffolds for fluorescence microscopic observation (n = 5) were fixed for an hour at 4% paraformaldehyde, treated an hour with 0.1% Triton-X, and stained with FITC-phalloidin (Sigma Aldrich, St. Louis, MO, USA) to show F-actin (red) + bisbenzimide H33342 fluorochrome trihydrochloride (Nacalai Tesque, Kyoto, Japan) and nuclei (blue) for an hour. After washing in PBS, they were observed with a fluorescence microscope (IX71, Olympus, Tokyo, Japan).
Scaffolds for SEM observation (n = 5) were fixed by 2.5% glutaraldehyde for 24 h, dried by 50–100% ethanol, sputter-coated by osmium, and observed by the FE-SEM (JEOL, Tokyo, Japan).

Cells cultured on Cellview glass bottom advanced TC 4 compartments (Griner Bio-one, Frickenhausen, Germany) were exposed to FT9110 for 48 h under the same conditions as described for the cell viability assay. For assessment, cells were stained with bisbenzimide H33342 fluorochrome trihydrochloride (H33342, 10 µg/mL; Nacalai Tesque, Kyoto, Japan) for 1 h before observation. The cells were visualized using an AxioObserverZ1 fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40× objective lens.