Under the same conditions described in Section 2.4 , the MC3T3-E1 cells were seeded and cultured in µ-Slide 8 wells (ibidi GmbH, Martinsried, Germany). The cells were rinsed twice with DPBS after 24 h of NFP exposure to NFPs in MC3T3-E1 cells (NFP-MC) and stained with nuclear dye (H33342 fluorescent dye trihydrochloride in dimethyl sulfoxide solution; Nacalai Tesque, Kyoto, Japan) and lysosome staining solution (CytoPainter Lysosomal Staining ab138895; Abcam, Cambridge, UK) for 30 min [26 (link)]. Subsequently, the cells were washed twice with DPBS, and images were captured using a BZ-X710 inverted fluorescence microscope (Keyence, Osaka, Japan).
Furthermore, after incubation under the aforementioned conditions for 24 h, the cells were trypsinized, fixed using a 4% paraformaldehyde phosphate-buffered solution (4% PFA; Nacalai Tesque), centrifuged at 300× g for 10 min at room temperature, and the 4% paraformaldehyde phosphate-buffered solution was subsequently removed. The cells were resuspended in DPBS, and the side scatter light (SSC) was evaluated using a BD FACSCelestaTM flow cytometer (FCM; BD, Franklin Lakes, NJ, USA) [27 (link),28 (link),29 (link)].
Furthermore, after incubation under the aforementioned conditions for 24 h, the cells were trypsinized, fixed using a 4% paraformaldehyde phosphate-buffered solution (4% PFA; Nacalai Tesque), centrifuged at 300× g for 10 min at room temperature, and the 4% paraformaldehyde phosphate-buffered solution was subsequently removed. The cells were resuspended in DPBS, and the side scatter light (SSC) was evaluated using a BD FACSCelestaTM flow cytometer (FCM; BD, Franklin Lakes, NJ, USA) [27 (link),28 (link),29 (link)].
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