N ethyl n 3 dimethylaminopropyl carbodiimide edc

The materials used in this study included glass substrates (7059 glass; Corning Inc., Corning, NY, USA), SiO₂ sputter target (purity ≥99.99%, THIFINE Co. Ltd., Incheon, Republic of Korea), indium tin oxide (ITO) sputter target (purity ≥99.99%, THIFINE Co. Ltd.), SnO₂ sputter target (purity ≥99.99%, THIFINE Co. Ltd.), 30:1 buffered oxide etchant (BOE; J.T. Baker, Phillipsburg, NJ, USA), phosphosilicate glass (PSG; Filmtronics Inc., Butler, PA, USA), polydimethylsiloxane (PDMS; Sylgard 184 silicon elastomer; Dow corning, Midland, MI, USA), pH buffer solution (Samchun chemical, Pyeongtack, Republic of Korea), ethanol (Samchun chemical), (3-aminopropyl)triethoxysilane (APTES; purity ≥99%, molecular weight = 221.37 g/mol, Sigma-Aldrich, St. Louis, MO, USA), 4-carboxyphenylboronic acid (4-CPBA; molecular weight = 165.94 g/mol, Sigma-Aldrich), N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC; purity ≥97%, molecular weight = 155.24 g/mol, Sigma-Aldrich), N-hydroxysuccinimide (NHS; purity ≥98%, molecular weight = 115.09 g/mol, Sigma-Aldrich), MES hydrate (purity ≥99.5%, molecular weight = 195.24 g/mol, Sigma-Aldrich), phosphate buffered saline (PBS; pH 7.4, Sigma-Aldrich), deionized water (DI water; conductivity ≤4.3 μS/cm, Sigma-Aldrich), and DA hydrochloride (gene information = ADRB1, molecular weight = 189.64 g/mol, Sigma-Aldrich).

Mouse monoclonal antibody against cTnT (mAb-cTnT), cTnT, and peroxidase conjugated mouse monoclonal antibody against cTnT (mAb-cTnT-HRP) were purchased from Calbiochem (Darmstadt, DEU). COOH-functionalized multiwalled carbon nanotubes (MWCNTs) were obtained from Dropsens (Oviedo, ESP). EDA, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), dimethylformamide (DMF), and glycine were acquired from Sigma-Aldrich (St. Louis, USA). Ethanol, sulfuric acid (H₂SO₄) (98%, w/v), and hydrogen peroxide (H₂O₂) (30%, w/v) were obtained from F. Maia (Cotia, BRA). All reagents were of analytical grade. The water used to prepare all solutions was obtained from a Milli-Q water purification system (Billerica, USA). All the solutions were freshly prepared prior to each experiment.

The following reagents were used in the experimental part: normal mouse IgG antibodies FITC conjugate (0.06 mg mL⁻¹) (SIGMA ALDRICH, Munich, Germany), anti-mouse IgG antibodies FITC produced in rabbit, IgG fraction of antiserum (10 mg mL⁻¹) (SIGMA ALDRICH, Munich, Germany), N-hydroxy-succinimide (NHS) (SIGMA ALDRICH, Munich, Germany), N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) (SIGMA ALDRICH, Munich, Germany), 11-Mercaptoundecanoic acid (MUA) acting as a linker (SIGMA ALDRICH, Munich, Germany), phosphate-buffered saline (PBS) pH = 7.4 (BIOMED, Lublin, Poland), acetate buffer pH = 3.50 (BIOMED, Lublin, Poland), carbonate buffer pH = 8.50–9.86 (BIOMED, Lublin, Poland). The solvents used to prepare solutions were absolute ethanol 99.8% (POCh, Gliwice, Poland) and MilliQ water (Simplicity^® Millipore, Burlington, MA, USA).
Measurements were performed with one type of glass chip coated with thin film metal layers: with a 0.1 nm of chromium, 44.8 nm layer of silver and a 3.3 nm layer of gold (Bialystok University of Technology).

Collagen fibers were used as a reinforcement for optimisation of mechanical properties of the collagen hydrogels. Fibers based on collagen (type I, VUP Medical, Brno, Czech Republic) were prepared via the electrospinning (4SPIN, Contipro, Dolní Dobrouč, Czech Republic) of an 8 wt% collagen PBS/ethanol solution modified by 8 wt% (to collagen) polyethylene oxide (PEO; Mr. 900,000, Sigma-Aldrich, St. Louis, MO, USA). The stability of all the collagen layers was enhanced by means of crosslinking with a 95% ethanol solution containing N-ethyl-N′-(3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a weight ratio of 4:1 (both Sigma-Aldrich, St. Louis, Missouri, USA), for 24 h at 37 °C. Cross-linked layers were further washed in 0.1 M Na₂HPO₄ (2 × 45 min), and by rinsed using deionised water (30 min). In this step, PEO was fully leached out [75 (link)]. The materials were then frozen at −30 °C for 5 h and lyophilised. Collagen fibers were then swelled in ethanol (100%), and their homogenisation, resulting in particle formation, was achieved using a disintegrator (10,000 rpm, 10 min), followed by rinsing with deionised water (30 min), freezing at −30 °C and lyophilising.

Collagenase type XI, exendin-4 (Ex4), Histopaque^®-1077, manganese (II) chloride (MnCl₂·4H₂O, 99%), iron (III) acetylacetonate (Fe(acac)₃, 99.9%), methyl poly(ethylene glycol) (mPEG, M.W. = 2000), N-hydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), oleic acid (90%), oleylamine (90%), osmium tetroxide (1%), propidium iodide (PI) and Prussian blue were purchased from Sigma–Aldrich (St. Louis, MO, USA). N-Boc-ethylenediamine (98%) and Acryloyl chloride (96%) were from Alfa Aesar (Ward Hill, MA, USA). Benzyl ether and (3-Aminopropyl) triethoxy silane (APTES, 98%) were from Fluka (Buchs, Switzerland). N-hydroxybenzotriazole (HOBt) and (Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP) were from NovaBiochem (Beeston, UK). RPMI-1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were from GIBCO BRL (Grand Island, NY, USA). Insulin radioimmunoassay (RIA) kit was from Millipore Corporation (Billerica, MA, USA). Polyethylene (PE-50) tubing was from Clay Adams (Parsippany, NJ, USA). Guinea pig anti-swine insulin antibody and rabbit anti-human glucagon antibody were from Dako (Carpinteria, CA, USA). Guinea pig anti-human insulin antibody (ab7842) was from Abcam (Cambridge, MA, USA).

Dicyclohexyl carbodiimide (DCC) (Cat # 802954), Dimethylformamide (DMF) (Cat # 103053), Poly (ethylene glycol) diacid (Cat # 843912), and anhydrous citric acid (Cat # 818707) were purchased from Merck. PD‐10 desalting columns containing Sephadex G‐25 resin (Cat # 170851) and Sephadex G15 (Cat # 170020) were purchased from GE Healthcare. N‐Hydroxysuccinimide (NHS) (Cat # 130672), RPMI 1640 (Cat # R8758), XTT (Cat # X4626), Hochest (Cat # B1155), and N‐Ethyl‐N′‐(3‐dimethylaminopropyl) carbodiimide (EDC) (Cat # E1769) were purchased from Sigma Aldrich. A549, L929, and NFS60 cell lines, rats, and mice were obtained from Iranian Genetic Resources Center and Pasteur Institute of Iran, respectively. G‐CSF was purchased from Cinnagen. Fetal bovine serum (FBS) (Cat # 10270) was purchased from GIBCO Invitrogen. Filter papers (Cat # DP 150‐150) and microplates (Cat # 3635) were purchased from Albet and corning, respectively. TSK G2000SWXL (Cat # 0008540), TSK G3000 SWXL (Cat # 0008541), and Guard (Cat # 0008543) were purchased from Tosoh. Quantikine ELISA kit for determination of G‐CSF (Cat # DCS50) was purchased from R&D systems.

Fibronectin from human plasma (lyophilised powder) as a standard, anti-fibronectin antibody produced in rabbit, collagen type IV, purified monoclonal mouse anti-human collagen type IV (Tebu-bio, Le Perray-en-Yvelines, France), bovine serum albumin (BSA), cysteamine hydrochloride, N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide (EDC) (Sigma-Aldrich, Steinheim, Germany), N-hydroxysuccinimide (NHS), (Sigma-Aldrich, Munich, Germany), photopolimer ELPEMER SD 2054, and hydrophobic protective paint SD 2368UV SG-DG (PETERS, Kempen, Germany) were used, as well as absolute ethanol, acetic acid, hydrochloric acid, sodium hydroxide, sodium chloride, sodium carbonate, sodium acetate, (POCh, Gliwice, Poland). HBS-ES buffer pH = 7.4 (0.01 M HEPES, 0.15 M sodium chloride, 0.005% Tween 20, 3 mM EDTA), Phosphate Buffered Saline (PBS) pH = 7.4, carbonate buffer pH = 8.5 (BIOMED, Lublin, Poland, http://www.biomed.lublin.pl) were used as received. Aqueous solutions were prepared using Milli-Q water (Simplicity^® Millipore).