Polyacrylamide gel substrates were prepared in accordance with previously published protocols70 (link),71 . In brief, the gel solution was prepared with 4% acrylamide, 0.1% bisacrylamide, 0.8% ammonium persulfate, 0.08% TEMED (Nacalai Tesque), and 5% deep red fluorescent carboxylate-modified beads (0.2 μm diameter; F8810; Thermo Fisher Scientific). In all, 13 μL of the mixture was added to a 35 mm glass-base dish (IWAKI) and then covered with a glass coverslip of 15 mm diameter (Matsunami). After gel polymerization at room temperature, the surface was coated with 0.3 mg/mL type I collagen (Nitta Gelatin, Osaka, Japan) using 4 mM sulphosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH; Pierce). Cells were seeded on the gel, and imaged with a spinning disk confocal microscope. To quantify the traction force, two Fiji/ImageJ (ver. 2.1.0/1.53c) plugins, i.e., the iterative PIV and FTTC plugins, were used. Note that Young’s modulus of the gel was estimated as ~2 kPa according to a previous report72 . The traction force in locally illuminated areas was used for the quantification.
Temed
Manufactured by Nacalai Tesque
TEMED (N,N,N′,N′-Tetramethylethylenediamine) is a chemical compound commonly used as a catalyst in gel electrophoresis techniques. Its primary function is to facilitate the polymerization of acrylamide, a crucial step in the preparation of polyacrylamide gels.
Automatically generated - may contain errors
2 protocols using temed
1
Traction Force Quantification on Collagen-Coated Gels
2
Polyacrylamide Gel Traction Force Assay
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Polyacrylamide gel substrates were prepared in accordance with previously published protocols (Tambe et al., 2011; Trepat et al., 2009) . In brief, the gel solution was prepared with 4% acrylamide, 0.1% bisacrylamide, 0.8% ammonium persulfate, 0.08% TEMED (Nacalai Tesque), and 5% deep red fluorescent carboxylate-modified beads (0.2 μm diameter; F8810; Thermo Fisher Scientific). 13 μL of the mixture was added to a 35 mm glass-base dish (IWAKI) and then covered with a glass coverslip of 15 mm diameter (Matsunami). After gel polymerization at room temperature, the surface was coated with 0.3 mg/mL type I collagen (Nitta Gelatin, Osaka, Japan) using 4 mM sulphosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH; Pierce). Cells were seeded on the gel, and imaged with a spinning disk confocal microscope. To quantify the traction force, two Fiji plugins, i.e., the iterative PIV and FTTC plugins, were used.
Note that Young's modulus of the gel was estimated as ~2 kPa according to a previous report (Tse and Engler 2010) . The traction force in locally illuminated areas was used for the quantification.
Note that Young's modulus of the gel was estimated as ~2 kPa according to a previous report (Tse and Engler 2010) . The traction force in locally illuminated areas was used for the quantification.
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