Cell morphology at Days 0, 1, and 3 was determined as described previously (Szot, Buchanan, Freeman, et al., 2011 (link)). Briefly, avascular platforms were fixed with 3.7% paraformaldehyde and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich). Then, samples were blocked with 1% BSA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 30 min at room temperature followed by an incubation step with rhodamine phalloidin (Invitrogen), a high-affinity probe for F-actin. Samples were counterstained with DAPI (Vector Laboratories, Burlingame, CA) to visualize nuclei. Imaging was performed using Leica TCS SP8 laser (Wetzlar, Germany) scanning confocal microscope. Another set of vascularized platforms were fixed to investigate cell morphology and ECM porosity using scanning electron microscopy (SEM). SEM preparation protocol is provided in the Supporting Information IV .
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Characterizing Cell Morphology and ECM Porosity
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Confocal microscope
Dapi
F actin
Nuclei
Paraformaldehyde
Rhodamine phalloidin
Scanning electron microscopy
Triton x 100
Vector
Corresponding Organization : The University of Texas at Austin
Other organizations : Dartmouth College, Livestrong Foundation
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Variable analysis
independent variables
- Days (0, 1, and 3)
- Cell morphology
- Fixation with 3.7% paraformaldehyde
- Permeabilization using 0.1% Triton X-100
- Blocking with 1% BSA for 30 min at room temperature
- Staining with rhodamine phalloidin and DAPI
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