The glass cover slips were treated with 3-aminopropyltrimethoxysilane and 0.5% glutaraldehyde. Then, 8% acrylamide (sigma, USA) was mixed with varying concentrations of bisacrylamide (0.1% and 0.7%) (Sigma, USA). Polymerization was initiated with N,N,N′,N′-tetramethylethylenediamine (TEMED) and ammonium persulfate (sigma, USA). Then, 0.2 mg/mL N-sulfosuccinyimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH) (Thermo, USA) dissolved in 10 mM HEPES (pH 8.5) was applied to cover polyacrylamide (PAAM) gel and exposed to 365 nm ultraviolet light for 70 min for photo activation in 24-well plates. The PAAm sheet was washed for three times with PBS to remove excess reagent and incubated with fibronectin solution (1 μg/cm2; Sigma, USA) overnight at 4° C. Before the cells were plated, the PAAm matrices were soaked in PBS and then in DMEM at 4° C. The Young’s modulus of the PAAm hydrogels was quantified using a biomechanical testing machine under contact load at a strain rate of 0.5 mm/s [45 (link)].
N sulfosuccinyimidyl 6 4 azido 2 nitrophenylamino hexanoate sulfo sanpah
Manufactured by Thermo Fisher Scientific
Sourced in United States
N-sulfosuccinyimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH) is a heterobifunctional cross-linking reagent. It contains an N-hydroxysuccinimide (NHS) ester for reaction with primary amines and an azido group for photoactivated conjugation to surfaces.
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3 protocols using n sulfosuccinyimidyl 6 4 azido 2 nitrophenylamino hexanoate sulfo sanpah
1
Substrate Preparation for Cell Culture
2
Tunable Polyacrylamide Substrates for Cell Culture
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Tunable polyacrylamide substrates were prepared as reported previously 16 (link). Briefly, glass coverslips were treated with 3-aminopropyltrimethoxysilane and 0.5% glutaraldehyde. Solution of 8% acrylamide (Sigma, USA) and varying concentrations of bis-acrylamide (0.1%, 0.5%, and 0.7%) (Sigma, USA) were mixed. Polymerization was initiated with N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (Sigma, USA). Then 0.2 mg/ml N-sulfosuccinyimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (sulfo-SANPAH) (Thermo, USA) dissolved in 10 mM HEPES (pH 8.5) was applied to cover the polyacrylamide gel and exposed to 365 nm ultraviolet light for 70 minutes for photoactivation in 24-well plates. The polyacrylamide sheet was washed three times with phosphate buffered saline (PBS) to remove excess reagent and incubated with fibronectin solution (1 μg/cm2; Sigma, USA) each well overnight at 4°C. Before cells were plated, the polyacrylamide substrates were soaked in PBS and then in DMEM at 4°C. The Young's modulus of polyacrylamide hydrogels was quantified using a biomechanical testing machine under contact load at a strain rate of 0.5 mm/s.
3
Tunable ECM Hydrogel Fabrication
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Tunable ECM was prepared based on a previous report [8 ]. Briefly, 8% acrylamide (Sigma-Aldrich, St. Louis, MO, USA) and varying concentrations of bis-acrylamide (0.1%, 0.3%, 0.5%, and 0.7%) (Sigma-Aldrich) were mixed and then polymerized with tetramethylethylenediamine (TEMED) and ammonium persulfate (AP) (Sigma-Aldrich) on aminosilanized 12-mm or 24-mm-diameter coverslips. We used previous methods to make ECM gels [32 (link)]. The polyacrylamide coverslips were subsequently coated with 0.2 mg/ml N-sulfosuccinyimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH; ThermoScientific, Waltham, MA, USA) dissolved in 10 mM HEPES (pH 8.5) and exposed to 365-nm ultraviolet light for 70 min. Subsequently, the coverslips were incubated in fibronectin solution (1 μg/cm2; Sigma-Aldrich, USA) overnight at 4 °C prior to cell plating. The elastic modulus for each concentration of polyacrylamide hydrogel was measured with a biomechanical testing machine under contact load at a strain rate of 0.5 mm/s.
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