Pbs ph 7

RI and LTI cells seeded on coverslips (Marienfeld GmbH & Co, Germany) were labelled for 30 min at 37°C with a 50 nM Lysotracker Red solution. Self-quenched BODIPY dye conjugated to bovine serum albumin labelling was performed (DQ-BSA; Molecular Probes, Invitrogen, USA), in order to measure lysosomal function. Cells were pre-incubated for 1 h at 37°C with 10 μg ml⁻¹ DQ-BSA in PBS. Then, cells were fixed at room temperature with 3% PFA in PBS pH 7.4 (Electron Microscopy Science, USA) for 10 min followed by 50 mM glycine in PBS pH 7.4 (Sigma, Germany) for 10 min.

MC-LR and MC-RR certified reference standards and MC-LF standard (stored at −20 °C), the anti-Adda antibody (AD4G2, stored at 4 °C), and the kit for MCs/nodularins PP2A (part number 520032, stored at 4 °C) were purchased from Eurofins Abraxis (Warminster, PA, USA). Ammonium bicarbonate, PBS (pH 7.4), and PBS containing Tween 20 (PBS-T) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure 18.2 MΩ-cm reagent-grade filtered water was produced in-house using an Aqua Solutions purification system (Jasper, GA, USA). Protein LoBind 2.0 mL microcentrifuge tubes, 96-well Protein LoBind 1.0 mL plates, 96-well TwinTec Lo-Bind PCR plates, and a Thermomixer C were purchased from Eppendorf (Hauppage, NY, USA). Acetonitrile was purchased from Pharmaco (Dawsonville, GA, USA). Methanol, formic acid, and adhesive PCR sealing foil sheets were obtained from Fisher Scientific (Waltham, MA, USA). Dynabeads MyOne Streptavidin T1, Zebaspin > 7 K molecular weight cut-off 0.5 mL desalting columns, and EZ-link NHS-PEG4-biotin were purchased from Life Technologies (Grand Island, NY, USA). A 96-well plate magnet was purchased from V&P Scientific (San Diego, CA, USA). A microcentrifuge tube magnet and a Dynabeads Sample Mixer were purchased from Invitrogen (Waltham, MA, USA).

A solution of PS spheres was purchased from Tianjin Dae Technology Co. 6‐Mercapto‐1‐hexanol (MCH) was purchased from Shanghai TCI Chemical Industry Co. Tris‐(2‐carboxyethyl)‐phosphine hydrochloride (TCEP) and ultrapure water (18.2 MΩ cm) were purchased from Shanghai Sheng Gong Co. Ltd. PBS (pH 7.4) and other chemicals were purchased from Sigma‐Aldrich; the DNA wash buffer contained 10  × 10⁻³m sodium citrate and 2% PEG, and the hybrid buffer contained 2% PEG and 10 × 10⁻³m PBS. All DNA single strand sequences were synthesized by Shanghai Sheng Gong Co. Ltd.; The DNA sequences are listed in Table S3.

All chemicals were reagent grade and used without further purification unless otherwise specified. Hexadecyltrimethylammonium bromide (CTAB; 99%), tetraethyl orthosilicate (TEOS; 99%), phosphate-buffered saline (PBS) pH 7.4 (0.01 M; with NaCl 138 mM and KCl 2.7 mM), and dialysis cellulose tubing membrane (3.5 kDa cut off) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triethanolamine (TEA; 99%) and sodium chloride (NaCl; 99.5%) were obtained from Fisher Chemical (Loughborough, UK). Trans-resveratrol (99%) was purchased from Tokyo Chemical Industry (TCI Europe N.V., Zwijndrecht, Belgium). Methanol (CH₃OH; >99%) and ethanol (CH₃CH₂OH; >99%) were acquired from VWR International (Radnor, PA, USA). All chemicals were used as received without further purification. Deionized water was used in all experiments and was produced using a Milli-Q system with a 0.22 μm filter (Synergy Equipment, Millipore, Burlington, MA, EUA).

Synthetic (Aib2) - oxyntomodulin (His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala, acetate salt, MW = 4447.9 g.mol^-1) (≥ 95% purity) and Synthetic native human oxyntomodulin (His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala, acetate salt, MW = 4449.9 g mol^-1) (≥ 95% purity) were purchased from Bachem (Switzerland). HPLC grade water (resistivity >18 MΩ cm), PBS (pH 7.4), phosphate, Tris-HCl, sodium dodecyl sulfate (SDS) and human collagen (Bornstein and Traub type I, recombinant, expressed in Nicotiana) were purchased from Sigma–Aldrich (UK). Sterile saline (0.9% (w v^-1) sodium chloride solution) (Baxter) and sterile water for injection BP (Fresenius Kabi) were purchased from VWR (UK). Millex syringe filters (pore size = 0.22 µm), Ultrafree-MC centrifugal filter devices with Durapore membranes (pore size = 0.22 µm) and Millipore Amicon Ultra-0.5 50K centrifugal filter devices were obtained from Fisher Scientific (UK). Hellmanex III was obtained from Hellma Analytics (Germany). Clear glass vials were obtained from MedImmune Nijmegen manufacturing facility (Netherlands).

Ovalbumin (OVA; grade V, ≥98% purity) and cow’s milk protein (CMP) rich in casein (87.1% [10 (link)]) were obtained from Sigma Chemical (Sigma-Aldrich, Saint Louis, MO, USA) and MP Biomedicals (Oklahoma, OH, USA), respectively. The test proteins were dissolved in PBS pH 7.4 (Sigma-Aldrich, Saint Louis, MO, USA; Cat: P3813), and the protein concentrations were determined (BCA assay, Thermo Scientific, Rockford, IL, USA).