Cells were mechanically preconditioned as indicated, and then plated onto 1.7 kPa or 8.5 kPa bead-embedded collagen I-coated hydrogels (30 μg/ml), prepared as detailed above. Imaging commenced 4 hours after plating onto bead-embedded hydrogels that were either 1.7 kPa or 8.5 kPa. Fluorescent microsphere beads (0.5 μm; Life Technologies) were dispersed throughout the hydrogels and excited with a red HeNe diode (561 nm) laser. PKH67-stained cells were visualized with an Argon (488 nm) laser. Three-dimensional image stacks were acquired using a Nikon A-1 confocal system mounted on a Ti-Eclipse inverted optical microscope controlled by NIS-Elements Nikon Software. A Plan Fluor 40X air 0.6 N objective (Nikon) mounted on a piezo objective positioner was used, which allowed imaging speeds of 30 frames per second using a resonant scanner. Confocal image stacks of 512 × 512 × 128 voxels (108 × 108 × 38 μm3) were recorded every 30 min with a z-step of 0.30 μm. Cell-induced full-field displacements were measured as previously described (Toyjanova et al., 2014 (link)) using the FIDVC algorithm (Bar-Kochba et al., 2015 ).
Fluorescent microsphere beads
Manufactured by Thermo Fisher Scientific
Fluorescent microsphere beads are small, spherical particles that emit light when exposed to a specific wavelength of light. These beads are commonly used in various scientific applications, such as flow cytometry, fluorescence microscopy, and cell labeling. The beads are available in a range of sizes and colors, allowing researchers to select the appropriate option for their specific needs.
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2 protocols using fluorescent microsphere beads
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Cell Mechanics on Tunable Hydrogels
2
Cell Mechanics on Tunable Hydrogels
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Cells were mechanically preconditioned as indicated, and then plated onto 1.7 kPa or 8.5 kPa bead-embedded collagen I-coated hydrogels (30 μg/ml), prepared as detailed above. Imaging commenced 4 hours after plating onto bead-embedded hydrogels that were either 1.7 kPa or 8.5 kPa. Fluorescent microsphere beads (0.5 μm; Life Technologies) were dispersed throughout the hydrogels and excited with a red HeNe diode (561 nm) laser. PKH67-stained cells were visualized with an Argon (488 nm) laser. Three-dimensional image stacks were acquired using a Nikon A-1 confocal system mounted on a Ti-Eclipse inverted optical microscope controlled by NIS-Elements Nikon Software. A Plan Fluor 40X air 0.6 N objective (Nikon) mounted on a piezo objective positioner was used, which allowed imaging speeds of 30 frames per second using a resonant scanner. Confocal image stacks of 512 × 512 × 128 voxels (108 × 108 × 38 μm3) were recorded every 30 min with a z-step of 0.30 μm. Cell-induced full-field displacements were measured as previously described (Toyjanova et al., 2014 (link)) using the FIDVC algorithm (Bar-Kochba et al., 2015 ).
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