Traction force microscopy with embedded beads was performed as described previously (Rafiq et al., 2019 (link)). Briefly, soft polydimethylsiloxanes CY 52-276A and CY 52-276B (Dow Corning, 0008602722) were mixed at the ratio 1:1 and Sylgard 184 crosslinker was used to tune the stiffness of the gel for proper force measurement of cells (∼95 kPa). The mixture was spin-coated onto a clean coverslip to achieve the thickness of ∼7 μm and cured for 1 h at 80°C. The surface of the gel was silanized with (3-aminopropyl)triethoxysilane for 2 h, followed by incubation of 0.04 μm FluoSpheres carboxylate-modified microspheres, dark red fluorescent (660/680) beads (Thermo Fisher Scientific, 1871942) at 1×106 beads/ml in a solution of 0.1 M NaHCO3 for 30 min. Before seeding the cells, the coverslips with beads were further incubated for 30 min with 10 μg/ml fibronectin, also dissolved in 0.1 M NaHCO3. Traction forces were calculated from bead displacement fields visualized by using live cell imaging as described by Tseng et al. (2012) (link), and using the online ImageJ plugin (https://sites.google.com/site/qingzongtseng/tfm for plugin software details). The computation algorithm as published by Sabass et al. (2008) (link) was used. The distribution of traction force magnitude was presented as a heat map (Fig. 1 A), and mean magnitude values were calculated for each cell.
Cy 52 276a
Manufactured by Dow
The CY 52-276A is a laboratory equipment product manufactured by Dow. It is designed for precise measurement and analysis tasks in scientific and research settings. The core function of this product is to facilitate accurate data collection and experimentation. No further details about its intended use or application are provided.
Automatically generated - may contain errors
Lab products found in correlation
Cy 52 276b, by Dow (6 mentions)
Glass bottom dishes, by MatTek (2 mentions)
Sylgard 184, by Dow (1 mentions)
Fluorescent beads, by Thermo Fisher Scientific (1 mentions)
Glass bottomed culture dishes, by MatTek (1 mentions)
Fluosphere, by Thermo Fisher Scientific (1 mentions)
Aptes, by Merck Group (1 mentions)
8 protocols using cy 52 276a
1
Traction Force Microscopy with Embedded Beads
2
Fabrication of Soft Elastomeric Silicone Gels
3
Fabrication of PDMS Substrates
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For PDMS substrates, 10:1, 20:1, and 50:1 ratios of PDMS to curing agent (Sylgard 184, Dow Corning) were mixed to generate 1.7 ± 0.2 MPa, 0.6 ± 0.5 MPa, and 50 ± 1.3 kPa, respectively. PDMS substrates (3 kPa) were fabricated by mixing silicone and curing agent (CY 52-276A and CY 52-276B, Dow Corning), as previously described (25 (link)). Elastomeric mixtures were placed into a vacuum desiccator for 2 min to remove air bubbles. Next, the mixed elastomeric solution was pipetted into each well of the tissue culture vessels. For imaging studies, a thin layer of PDMS was coated onto a 35-mm, 14-mm MatTek No. 1.0 cover glass, using a speed of 1000 rpm for 60 s with a 100-rpm acceleration time to spin-coat PDMS substrates. The spin-coated dish was placed on a flat surface and cured for 30 min at 70°C for 3-kPa substrates and for 1 hour at 65°C for the Sylgard 184 surfaces.
4
PDMS Gel Substrate Preparation
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Round glass bottom dishes (Willco Wells) were coated with PDMS silicone gel: a 1:1 mixture of components CY 52-276-A and CY 52-276-B (Dow Corning Toray). After being degassed, the mixture was pipetted onto the dishes and spin-coated to the desired thickness of approximately 20 μm. The mixture was cured overnight at room temperature (RT) to obtain a gel with Young’s modulus 3 KPa22 (link). To quantify deformation, the gel contained two layers of fluorescent beads (radius 100 nm, Invitrogen): one stationary layer between the glass and gel, and one layer around 20 μm above the dish, which moved in response to cellular forces. PDMS-coated dishes were soaked in 70% ethanol for 10 minutes and air-dried in a biosafety hood to sterilize before cell plating. Substrates were then coated with Matrigel, and cell culture was performed as described above.
5
Soft 3 kPa PDMS Gel Fabrication
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The soft 3 kPa PDMS gels were prepared by mixing a 1:1 ratio (w/w) of CY 52-276A and CY 52-276B (Dow Corning). Components were mixed thoroughly and immediately degassed for 15 min in a desiccator. The surface of 35 mm glass-bottomed culture dishes (MatTek Corporation) were covered with 200 µL of the mixed gel. Each dish was then spin-coated at 1000 rpm for 60 s using a spin coater instrument (Headway Research, PWM32). Gels were cured at room temperature overnight. The next morning gels were sterilized for 30 min under UV light and then washed once with sterile PBS. Rheometry analysis (ARES-LS1) confirmed that the Young’s modulus of the PDMS was ~3 kPa.
6
Preparation of Soft PDMS Gel Substrates
7
Fabrication of Soft Silicone Gel Substrates
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Soft silicone gels were prepared using a protocol based on previous publications55 (link)–58 (link). Briefly, the silicone elastomer was synthesized by mixing a 1:1 weight ratio of CY52-276A and CY52-276B polydimethylsiloxane (Dow Corning Toray). After degassing for 30 min in ice, the gel was spin-coated on glass-bottom dishes (35-mm, no. 0 coverslip thickness, Mattek) for 90 s at 400 r.p.m. The samples were then cured at 65 °C overnight. The substrates were kept in a clean and dry environment and they were used within 8 weeks of fabrication.
8
Functionalizing Polyacrylamide Gels with Nanobeads and Fibronectin
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Polyacrylamide gels with a Young's modulus of 15 kPa were prepared using CY-52-276 A and CY-52-276 B (Dow Corning Toray) in 1:1 v/v ratio. Gels were spin coated at 500 RCF for 30 secs to get an even coating and cured for 2 hours at 80°C. 5% APTES (Sigma-Aldrich A3648) in ethanol was added to the gels followed by an incubation of 10 minutes. The solution was then removed, and the gels were washed with ethanol and dried at 80°C for 30 minutes. 200 nm nanobeads (Thermo Fisher Scientific, FluoSpheres®) were diluted at 1:30000 with distilled water and sonicated for 10 minutes to break the clumps. The silane-PDMS was functionalised with the nanobeads solution for 5 minutes at room temperature after which the beads were removed again. Functionalised gels were rinsed with distilled water and dried for 15 minutes at 80°C. Beads were then passivated with 100 mM Tris for 10 min at RT. Subsequently, the solution was removed, gels were washed with distilled water and dried in the oven for 15 minutes at 80°C. 10 g/ml fibronectin was added to the gels followed by 1 hour incubation at room temperature. Next, the gels were incubated with 1% Pluronic for 30 mins to sterilise and washed with PBS. Finally, cells were added and monitored until a confluent monolayer was generated.
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