To facilitate cell adhesion to PA gels, the bifunctional crosslinker sulfo-SANPAH was deposited onto the gel surface, enabling subsequent collagen I conjugation. PBS was removed from the PA gels in 24-well plates and a solution of 1 mg.mL−1 sulfo-SANPAH (ThermoFisher Scientific) in deionized water was added on top of each gel to yield 25 μg.cm−2 sulfo-SANPAH. Next, the plate was exposed to UV light for 15 minutes for photoactivation of the crosslinker, the darkened sulfo-SANPAH solution was removed, and the process was repeated for a second coating of sulfo-SANPAH. After the second deposition, sulfo-SANPAH was removed and the wells were washed several times in 1X PBS to remove unreacted sulfo-SANPAH. PA gels were then incubated with a dilute solution of rat tail collagen I (Fisher Scientific) in 0.02 N acetic acid overnight at 4°C, to yield a final protein coating of 5 μg.cm−2. The next day, PA gels were washed twice with 1X PBS to remove residual collagen I and acetic acid.
Sulfo sanpah
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Sulfo-SANPAH is a heterobifunctional crosslinking reagent used for covalent immobilization of proteins and other biomolecules onto surfaces. It contains a sulfo-N-hydroxysuccinimide (sulfo-NHS) ester group and a photoreactive phenylazide group, allowing for two-step attachment of molecules to various substrates.
Automatically generated - may contain errors
Lab products found in correlation
Temed, by Merck Group (8 mentions)
Acrylamide, by Bio-Rad (7 mentions)
3 aminopropyltrimethoxysilane, by Merck Group (7 mentions)
Ammonium persulfate, by Merck Group (7 mentions)
Bis acrylamide, by Bio-Rad (6 mentions)
Glutaraldehyde, by Merck Group (6 mentions)
Ammonium persulfate, by Bio-Rad (5 mentions)
Fibronectin, by Merck Group (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
3 aminopropyltriethoxysilane, by Merck Group (4 mentions)
Acrylamide solution, by Bio-Rad (4 mentions)
Ammonium persulfate, by Thermo Fisher Scientific (3 mentions)
Aminopropylsilane, by Merck Group (3 mentions)
Glutaraldehyde, by Electron Microscopy Sciences (3 mentions)
Rat tail collagen type 1, by Corning (3 mentions)
Collagen 1, by Merck Group (3 mentions)
N n methylene bis acrylamide solutions, by Bio-Rad (3 mentions)
Fibronectin, by Thermo Fisher Scientific (2 mentions)
Acrylamide bis solution, by Bio-Rad (2 mentions)
Anti cam, by Merck Group (2 mentions)
96 protocols using sulfo sanpah
1
PA Gel Surface Functionalization for Cell Adhesion
2
Polyacrylamide Gel Synthesis and Functionalization
3
Polyacrylamide Gel Substrate Preparation
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The confocal dishes were coated with a 1.0 kPa polyacrylamide (PA) gel with stiffness much lower than that of glass67 (link). Briefly, Confocal dishes were rinsed with 0.1 M NaOH, dried, incubated for 5 min with 3-aminopropyltriethoxysilane, washed with distilled water, incubated with 0.5% glutaraldehyde for 30 min, and air-dried. The PA-gel solution was then added to the dishes such that the gel’s final thickness would be around 50 μm, and a diameter of 12 mm coverslip was gently laid on it. The solution contained APS, TEMED, acrylamide, and bis-acrylamide (all from Pierce Biotechnology) at a ratio such that the polymerized gel would have a stiffness of 1.0 kPa. The gels were allowed to polymerize, the coverslips removed, and the dishes were thrice washed with PBS. The gels were then functionalized by adding a sulfo-SANPAH (Pierce Biotechnology) solution and exposing them to UV light of 365 nm for 15 minutes to covalently link the gel to the sulfo-SANPAH. Next, the dishes were sterilized by UV light in a cell culture hood for 30 min and then coated with poly-L-lysine (PLL, Gibco) for the following cell seeding.
4
Polyacrylamide Gel Fabrication and Functionalization
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PA gels were generated as previously described (Simi et al., 2018 (link)). Briefly, PA gels were polymerized on 31-mm-diameter glass coverslips by mixing water and 12.5% (vol/vol) acrylamide with either 0.5% (vol/vol) or 17.5% (vol/vol) bisacrylamide. To initiate polymerization, 10% ammonium persulfate (Bio-Rad) at a 1:200 dilution and N,N,N’,N’-tetramethethylenediamine (Sigma-Aldrich) at a 1:2000 dilution were added; 36 μl of the mixture was sandwiched between the coverslips, and the mixture was allowed to polymerize for 45 min at room temperature, after which the top coverslip was carefully removed. The heterobifunctional crosslinker Sulfo-SANPAH was used to conjugate the surface of the PA gel with fibronectin (Fisher Scientific) as previously described (Simi et al., 2018 (link)). Before cells were plated, the gels were rinsed at least twice with HEPES buffer or phosphate-buffered saline (PBS) and incubated with culture medium at 37°C for ∼1 h.
5
Collagen Protein Immobilization on Hydrogels
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Gels were immersed in 10 mg/ml sulfo-SANPAH (Fisher Scientific) in 50 mM, pH 8.5, HEPES and reacted under 365 nm i-line exposure for 10 min. Collagen was mixed in 0.1 M acetic acid (Fisher Scientific) at equal volume and in 50 mM, pH 8.5, HEPES to reach 0.2 mg/ml final concentration. Gels were immersed with collagen while agitated overnight at 37°C. Prior to seeding cells, gels were UV-sterilized (cell-culture hood UV light source) for 3 h. During all preparation steps, gels were maintained in a hydrated state.
6
Acrylamide Hydrogel Preparation Protocol
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Acrylamide hydrogel was prepared according to published protocols81 . Briefly, glass-bottomed dishes were first cleaned and activated with 1% v/v (3-Aminopropyl) triethoxysilane (APTES, Sigma–Aldrich) in ethanol. The silanized coverslips were baked at 65 °C overnight. Next the glass surface was treated with 0.5% v/v glutaraldehyde in Milli-Q water for 30 min, then cleaned and dried. The gel solution (acrylamide, bis-acrylamide) in phosphate buffered saline (PBS) was first degassed in a vacuum desiccator for 1 h. After that, 10 μl of 10% w/v ammonium persulfate (APS, Sigma–Aldrich) and 1 μl of N,N,N′,N′-Tetra-methyl-ethylenediamine accelerator (TEMED, Sigma–Aldrich) per ml of solution was added. After mixing, the solution was poured onto the glass top and covered with a coverslip. After 30 min of incubation at RT, coverslips were removed, and dishes were washed three times with PBS. The gel was then incubated with 0.5% Sulfo-Sanpah (Fisher) in water and exposed to UV light (UV cross linker) for 10 min. After repeating incubation and UV exposure, the dishes were washed and incubated in a fibronectin solution (0.1 mg/ml) overnight. Cells were seeded the next day after thoroughly cleaning the dishes.
7
Preparation of Tunable PA Hydrogels for Cell Culture
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Cover glass (25 mm or 12 mm) were cleaned with plasma cleaner for 2.5 min and the glass surface was treated with APTES (VWR) for 5 min. The glass coverslips were washed in DI water 2-3 times, soaked in glutaraldehyde (Electron microcopy Solution, EMS) for 30 min at RT in a fume hood, washed once with DI water, dried and stored in a dessicator until used. PA hydrogels (1, 10 and 100 kPa) were prepared by mixing 40 % acrylamide and 2 % bis -acrylamide solution (Biorad) followed by addition of APS and TEMED (VWR) for gel polymerization [65, 66] . A droplet of gel was placed on a cover glass which was flipped over a glass slide to form a gel "sandwich". Gels were polymerized for 30 min, washed with PBS 2-3 times and stored at 4 o C in PBS no longer than 7 days. On the day of cell seeding, gels were washed with 50mM HEPES (pH-8.5) for 2-3 times and incubated with 0.2 mg/ml Sulfo-SANPAH (Fisher Scientific). After Sulfo-SANPAH treatment, gels were immediately placed under 365 nm UV light source at a distance of approximately 3 inches and exposed for 10 min. Gels were washed with HEPES for 2-3 times and incubated with 20 µg/ml of fibronectin for minimum 3 h at 37 o C. Gels were finally washed with PBS twice and used as a substrate for cell plating.
8
Quantifying CaM Binding to RyR2 Using Photoreactive Cross-Linking
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Binding of CaM to RyR2 was evaluated using the photoreactive cross-linker, sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino]hexanoate (Sulfo-SANPAH, Thermo Fisher Scientific, Waltham, USA), as described previously11 (link). First, we made a CaM-SANPAH conjugate by mixing 50 μM recombinant CaM in conjugation buffer (150 mM KCl and 20 mM MOPS at pH 7.2) with 100 μM Sulfo-SANPAH in the dark for 30 min. Conjugation was quenched by adding excess amount of lysine. CaM-SANPAH conjugate was purified using Amicon Ultra (MWCO 10 k). Mouse brain homogenates were diluted in binding buffer (150 mM KCl, 10 μM CaCl2, and 20 mM MES at pH 6.8) to 1 mg/mL and mixed with 100 nM CaM-SANPAH conjugate in the dark in a glass tube with or without CaMBPs. After a 10-min binding time, 30 s UV crosslinking was performed. Then, sample buffer was added to the crosslinked SR membrane followed by western blotting with anti-CaM (Merck, Millipore, Darmstadt, Germany). CaM-SANPAH crosslinked to RyR2 was detected as a 550 kDa band.
9
Polyacrylamide Hydrogel Preparation and Functionalization
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Polyacrylamide hydrogels (PAGs) were cast onto No. 0 glass bottom 35 mm culture dishes (MatTek P35G-0-20-C) using a previously described method and formulation [13 (link), 51 (link)]. Cast gels were then coated using sulfo-SANPAH (Thermo Fischer Scientific, Waltham, MA) as previously described (Bangasser et al. [13 (link)], Wang and Pelham [51 (link)]). In this study, Type I Collagen (354236, Corning, Corning, NY) or anti-CD44 antibody α-CD44 MAB (BDB553131, BD Biosciences, San Jose, CA) were used to coat PAGs. In general, 200 μg/mL Col I solution was used and 1-300 μg/mL α-CD44 MAB solution was used depending on the experiment. The Young’s Modulus of the PAGs was determined using the methods described by Bangasser et al.. 0.7, 4.6, 9.3, 19.8, 98.5, 195 kPa stiffnesses were used in this study, which were obtained by varying the cross-linker and polymer concentrations as previously described [13 (link)].
10
Substrate Preparation for Cell Culture
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Glass or polystyrene culture dishes were coated with 10‐µg/ml fibronectin with or without the addition of 50‐µg/ml TTR fibrils or native tetramer in phosphate‐buffered saline (PBS) solution. Dishes were coated for 2 h in a 37°C, 5% CO2 incubator before cell plating.
Microgrooved substrates were prepared by molding 400 kPa polydimethylsiloxane (PDMS) from a parylene template to produce spaced grooves as done previously, with grooves 10‐µm wide, 5‐µm high, and ridges 10‐µm wide (Motlagh, Senyo, et al., 2003 (link)). Before coating with fibronectin or TTR fibrils, PDMS microgroove substrates were functionalized with 3‐aminopropyl triethoxysilane (Sigma‐Aldrich cat# 440140).
Polyacrylamide (PAA) substrates with a stiffness of 10 kPa were prepared as previously described (Li et al., 2016 (link)). Before polymerization, fluorescent microspheres (Invitrogen cat# F8807) were included in substrates for later traction force microscopy (Broughton et al., 2016 (link); Ribeiro et al., 2017 (link)). PAA substrates were functionalized with Sulfo‐Sanpah (Thermo Fisher cat# 22589) before coating with fibronectin or TTR fibrils.
Microgrooved substrates were prepared by molding 400 kPa polydimethylsiloxane (PDMS) from a parylene template to produce spaced grooves as done previously, with grooves 10‐µm wide, 5‐µm high, and ridges 10‐µm wide (Motlagh, Senyo, et al., 2003 (link)). Before coating with fibronectin or TTR fibrils, PDMS microgroove substrates were functionalized with 3‐aminopropyl triethoxysilane (Sigma‐Aldrich cat# 440140).
Polyacrylamide (PAA) substrates with a stiffness of 10 kPa were prepared as previously described (Li et al., 2016 (link)). Before polymerization, fluorescent microspheres (Invitrogen cat# F8807) were included in substrates for later traction force microscopy (Broughton et al., 2016 (link); Ribeiro et al., 2017 (link)). PAA substrates were functionalized with Sulfo‐Sanpah (Thermo Fisher cat# 22589) before coating with fibronectin or TTR fibrils.
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