The spheroid formation assay was performed on 1% agarose-coated culture plates. Ten thousand cells/mL were cultured in media supplemented with the LC50 of Cc-AgNPs for 10 days. The morphology and size of spheroids were observed through the Olympus fluorescence microscope software cellSens (Ver.1.18, Wetzlar, Germany).
Cellsens ver 1
Manufactured by Olympus
Sourced in Germany, Japan
CellSens (Ver.1.18) is a software application developed by Olympus for image acquisition, processing, and analysis of microscopic samples. The software provides core functionalities for controlling microscope hardware, capturing images, and performing basic image analysis tasks.
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2 protocols using cellsens ver 1
1
Spheroid Formation Assay with Cc-AgNPs
2
Morphological Characterization of A-MSCs
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Monolayer and 3D spheroid A-MSCs were subjected to scanning electron microscopy by WeiYa Bio-Technology Co., Ltd. (Jinan, Shandong, China). Samples were fixed using 2.5% glutaraldehyde solution for 3 h at 4°C. Microstructural characterization was performed using a high-resolution scanning electron microscope (Sigma300; Zeiss, Germany).
To observe the overall distribution of cells, digested A-MSCs were first stained with PKH26 using PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich, St Louis, MO, USA). Prior to staining, PKH26 in diluent C was incubated in a water bath at 37 °C for 15 min. A-MSCs stained with a cell membrane were cultured by the hanging drop method. After 24 h incubation in a 37°C incubator, the original position of the suspension was maintained for 4′,6-diamidino-2-phenylindole (DAPI) staining. Next, 3D spheroid A-MSCs were subjected to hematoxylin and eosin staining for morphological evaluation. The spheroids were observed under a microscope (BX53; Olympus, Tokyo, Japan) and photographed (CellSens, Ver. 1.18; Tokyo, Japan).
To observe the overall distribution of cells, digested A-MSCs were first stained with PKH26 using PKH26 Red Fluorescent Cell Linker Kits for General Cell Membrane Labeling (Sigma-Aldrich, St Louis, MO, USA). Prior to staining, PKH26 in diluent C was incubated in a water bath at 37 °C for 15 min. A-MSCs stained with a cell membrane were cultured by the hanging drop method. After 24 h incubation in a 37°C incubator, the original position of the suspension was maintained for 4′,6-diamidino-2-phenylindole (DAPI) staining. Next, 3D spheroid A-MSCs were subjected to hematoxylin and eosin staining for morphological evaluation. The spheroids were observed under a microscope (BX53; Olympus, Tokyo, Japan) and photographed (CellSens, Ver. 1.18; Tokyo, Japan).
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