Substrates for TFM were prepared from soft silicone gels16 (link)39 (link). Briefly, soft silicone gels were mixed in a ratio of 1:1 and spin coated on a glass bottom Petri dish and cured. The gels were silanized by incubating in 5% (3-aminopropyl)triethoxysilane (APTES, Sigma) in ethanol for 5 min. The substrates were dried and incubated with 100 nm carboxylated fluorescent beads (Invitrogen) for 5 min, washed and dried. The beads were passivated with 100 mM Tris base (1st Base) in water and dried. Fibronectin pattern was stamped on the soft silicone gels using a water-soluble polyvinyl alcohol (PVA) membrane as an intermediate substrate31 (link). The gels were finally blocked with 0.2% Pluronics solution. Cells were seeded on the patterns and imaged on an Olympus inverted microscope. After imaging, cells were lysed and the ‘stress free’ image of the beads was obtained. The unconstrained stresses were computed using custom written MATLAB codes32 (link).
Carboxylated fluorescent beads
Manufactured by Thermo Fisher Scientific
Carboxylated fluorescent beads are spherical particles that emit fluorescent light when exposed to specific wavelengths of light. These beads are surface-functionalized with carboxyl groups, allowing for covalent attachment of biomolecules such as proteins, peptides, or nucleic acids. The fluorescent properties of these beads make them useful for various applications in biotechnology and life sciences.
Automatically generated - may contain errors
3 protocols using carboxylated fluorescent beads
1
Traction Force Mapping on Silicone Gels
2
Traction Force Microscopy with Soft Silicone Gel
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Soft silicone gel attached with fluorescent beads was used as substrate for TFM so that in-plane cell traction forces on the substrate could be measured35 . To prepare the gel with a stiffness of 10 - 20 kPa, CyA and CyB components (Dow Corning) were mixed at 1:1 ratio and spin-coated on a petri-dish to achieve a flat substrate of height ˜ 60 - 100 μm. After curing at 80°C for 2hr, the substrate was silanized with 5% (3-Aminopropyl) trimethoxysilane (Sigma) in ethanol for 5 min. Carboxylated fluorescent beads (100 nm, Invitrogen) were functionalized on the substrate at 1:500 dilution in DI water. The beads were passivated with 1X Tris (Sigma) for 10 min and pure fibronectin (50 μg/ml) was incubated on the substrate for 1 hr prior to cell seeding. Between each step, the samples were rinsed 3 times with 1X PBS. After the experiment, the cells were completely removed by adding SDS, such that the resting state of the gel can be measured. Bead displacements (with respect to its resting state) acquired during experiments were measured and converted to cell traction forces with an ImageJ plugin36 .
3
Traction Force Microscopy with Soft Silicone Gel
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