Confocal microscopy was performed to visualise the morphology of the cells attached onto the scaffolds cultured in basal medium after 7 and 14 days. Each scaffold was rinsed twice in PBS (Polysciences, Warrington, PA, USA) and fixed in 4% (w/v) paraformaldehyde in PBS with 1% (w/v) sucrose at 37 °C for 4 h. Thereafter, the scaffolds were rinsed twice with PBS and then the samples were incubated 5 min with Triton 0.1% (w/v) at room temperature to permeabilise the cells. Then, the scaffolds were rinsed with PBS and incubated with 400 μL, 6-diamidino-2-phenyl-indole (DAPI) (DAPI, Vector Laboratories, Burlingame, CA, USA) (1:1000) and Atto 565-conjugated phalloidin at a concentration of 0.165 μM (Molecular Probes) for 30 min. DAPI (cell nuclei) and phalloidin (cytoskeleton) were visualised in blue and red, respectively.
6 diamidino 2 phenyl indole dapi dapi
Manufactured by Vector Laboratories
Sourced in United States
DAPI (6-diamidino-2-phenyl-indole) is a fluorescent stain that selectively binds to DNA. It is commonly used in fluorescence microscopy to visualize and identify cell nuclei.
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2 protocols using 6 diamidino 2 phenyl indole dapi dapi
1
Visualizing Cell Morphology on Scaffolds
2
Visualizing Encapsulated Spheroid Morphology
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Confocal microscopy was performed to visualise the morphology of the GeMAencapsulated spheroids injected into the scaffolds at 1, 4, 7, and 14 and 21 days (n=2).
Each scaffold was rinsed twice in phosphate buffer solution (PBS) and fixed in 4% (w/v) paraformaldehyde in PBS at 37 °C for 4 hr. Then, the samples were incubated for 5 min with Triton 0.1% (w/v) at room temperature to permeabilise the cells. After, the scaffolds were rinsed with PBS and incubated with 400 μL, 6-diamidino-2-phenylindole (DAPI) (DAPI, Vector Laboratories, Burlingame, CA, USA) (1:1000) and Atto 565-conjugated phalloidin at a concentration of 0.165 μM (Molecular Probes) for 30 min and subsequently imaged using a Nikon Confocal Microscope (Eclipse-Ti, U.S.A).
Each scaffold was rinsed twice in phosphate buffer solution (PBS) and fixed in 4% (w/v) paraformaldehyde in PBS at 37 °C for 4 hr. Then, the samples were incubated for 5 min with Triton 0.1% (w/v) at room temperature to permeabilise the cells. After, the scaffolds were rinsed with PBS and incubated with 400 μL, 6-diamidino-2-phenylindole (DAPI) (DAPI, Vector Laboratories, Burlingame, CA, USA) (1:1000) and Atto 565-conjugated phalloidin at a concentration of 0.165 μM (Molecular Probes) for 30 min and subsequently imaged using a Nikon Confocal Microscope (Eclipse-Ti, U.S.A).
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