To recover cells from the 3D constructs, the scaffolds were disaggregated into 1–2 mm3 pieces with sterile surgical blades and enzymatically digested in 2 mg/ml type I collagenase (Merck Millipore, Darmstadt, Germany) for 1 h at 37 °C in stirring conditions. Cell suspension was then filtered with 100 µm sterile CellTrics (Partec, Münster, Germany). For morphological evaluation, recovered cells were plated in 4-multiwell chamber slides. After 24 hours cells were fixed and stained for Phalloidin and DAPI (Invitrogen) and analyzed though fluorescence inverted microscopy (Nikon, Tokyo, Japan).
Phalloidin and dapi
Manufactured by Thermo Fisher Scientific
Sourced in Japan
Phalloidin is a bicyclic heptapeptide that binds specifically to F-actin, a structural component of the cytoskeleton. DAPI is a fluorescent stain that binds strongly to the A-T rich regions in DNA. These molecules are commonly used in microscopy techniques to visualize and study cellular structures and processes.
Automatically generated - may contain errors
2 protocols using phalloidin and dapi
1
3D Scaffold Cell Recovery Protocol
2
Morphological Analysis of 3D Cell Cultures
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To assess the morphology of cells after 3D culture with scaffolds, MC3T3‐E1 and Raw264.7 cells were selected to be inoculated on the surface of porous samples for culture, respectively. After 1 or 3 days of cell‐scaffold co‐culture, cells were lysed with 1% Triton X‐100 (Beyotime, Shanghai, China) followed by Rhodamine‐Phalloidin staining (Invitrogen). Phalloidin and DAPI (Invitrogen) were used to label the cytoskeleton and nuclei. They were then visualized using a CLSM. In addition, co‐cultured cells were fixed with glutaraldehyde, dehydrated with gradient alcohol, and examined using SEM.
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