HUVECs (cat. no. #8000; ScienCell Research Laboratories, Inc., Carlsbad, CA, USA) were collected as described previously (26 (link)). They were cultured in HuMedia supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), 10 ng/ml recombinant epidermal growth factor, 1 ng/ml hydrocortisone, 5 ng/ml recombinant fibroblast growth factor and 10 ng/ml heparin under 5% CO2 at 37°C. HUVECs were incubated in a normal glucose concentration (1,000 mg/l, group N), or high glucose concentration (4,000 mg/l, group D) without or with MCPs [3.0 mg/ml (group L), 15.0 mg/ml (group M) or 30.0 mg/ml (group H)] for 24, 48 or 72 h. The dose of MCPs in group H was calculated based on the following formula: typical clinical dose (2.25 g/kg) × standard adult male BW (60 kg)]/average blood volume (4,500 ml). The doses in groups M and L were one-fifth and one-tenth those in group H, respectively. Cholecystokinin-octopeptide assays were performed to assess cell growth inhibition using a cell counting kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), which were detected using an ELISA microplate reader (LabSystems Multiskan MS 352; Thermo Fisher Scientific, Inc.), and the growth inhibition ratio was calculated using the formula: Growth inhibition ratio=[normal control optical density (OD) values-treatment group OD values]/normal control OD values ×100%.
Labsystems multiskan ms 352
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States, Finland, China
The Labsystems Multiskan MS #352 is a compact, single-beam photometer used for microplate-based absorbance measurements. It is designed to perform basic absorbance-based assays, such as ELISA, in 96-well microplates.
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6 protocols using labsystems multiskan ms 352
1
Effect of MCPs on HUVEC Growth
2
Serum Biomarker ELISA Measurement
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The serum samples ELISA measurement was conducted according to commercial ELISA kits instructions to determine the pharmacological indexes of PRA, Ang I, Ang II, and ALD. The absorbance of each sample was measured on a Lab systems Multiskan MS 352 microreader (Thermo Fisher Scientific Inc., USA). The concentrations of PRA, Ang I, Ang II, and ALD were calculated based on each calibration curve, respectively.
3
Quantification of Alkaline Phosphatase Activity
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ALP activity was assayed using LabAssay™ ALP according to the manufacturer’s protocol (#291–58601, Wako Pure Chemical Industries, Ltd.). Briefly, three control, three PXE, and one GGCX fibroblast cell lines were seeded in triplicate (1.6 × 105 per well in a 48-well plate) and underwent osteogenic induction for 3 days starting the next day. Thereafter, cells were lysed by two freeze-thaw cycles with 150 μl of 0.05% Triton X-100 prepared in buffer (2.0 mmol/L MgCl2 and 0.1 mol/L carbonate buffer, pH 9.8). After centrifugation at 15,000 rpm for 15 minutes, supernatants (20 μl) were incubated for 25 minutes with ALP substrate (6.7 mmol/L p-nitrophenylphosphate disodium). Reactions were stopped by addition of stop solution (0.2 mol/L sodium hydroxide solution), followed by measurement of OD at 405 nm in triplicate (Labsystems Multiskan MS #352, Thermo Fisher Scientific Inc.). The values were adjusted by cell numbers, which were obtained by analysis of cells seeded in parallel.
4
Quantifying ucMGP in GGCX Fibroblasts
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ucMGP concentrations in GGCX dermal fibroblasts were quantified using a human ucMGP ELISA Kit according to the manufacturer’s protocol (#CSB-EC013789HU, Cusabio Biotech Co., Wuhan, China). Briefly, cells (6.4 × 105) isolated from three controls and one GGCX patient were separately seeded on 6-well plates. When they reached early confluency, cells were washed with cold PBS twice and lysed on ice for 5 minutes with RIPA buffer (#9806S, Cell Signaling Technology) containing protease inhibitors (Protease Inhibitor Cocktail Set I; Calbiochem) and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz). The lysate were collected using a cell scraper and vortexed for 30 seconds. After centrifugation at 12,500 rpm for 10 minutes at 4°C, protein concentrations in the supernatants were quantitated using a Quick Start Bradford protein assay (Quick Start BSA standard set, Bio-Rad Laboratories, Inc.). Samples (approximately 40 μg of protein) were mixed with the sample diluent, and the ucMGP content was assessed. Reactions were stopped by addition of acidic stop solution, followed by measurement of OD at 450 nm in triplicate (Labsystems Multiskan MS #352, Thermo Fisher Scientific Inc.). The values were adjusted by the protein contents, which were determined from the same plates.
5
Quantification of Plant Hormones in Berries
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For the quantification of auxin, cytokinin, gibberellin and brassinosteroid, 1 g of fresh berries was ground in a mortar and homogenized in extraction solution (9 ml of PBS, pH 7.2–7.4) for enzyme-linked immunosorbent assay (ELISA) [49 (link)]. The ELISA procedures were conducted according to the instructions provided by the manufacturer (Enzyme-linked biological technology Co., LTD., Shanghai, China) on the Thermo Labsystems AC8 and Labsystems Multiskan MS 352 (Thermo Labsystems Co., Vantaa, Finland).
6
Serum Cytokine Evaluation Protocol
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After the intervention and all other assessments had been completed, a 3-mL sample of blood was obtained from each subject the next morning between 7:30 a.m. and 8:00 a.m., with fasting and avoidance of strenuous activity and mental stimulation. On this condition, all serum cytokines measured showed stable values according to each individual’s condition. The sample was allowed to stand at room temperature for 30 min and then centrifuged at 3000 rpm for 5 min; the serum was stored with anticoagulant and frozen at −80°C. TGF-β1, GM-CSF, IFN-γ, and RANTES levels in the serum were measured using enzyme-linked immunosorbent assay kits (Shanghai Jianglai Co, Shanghai, China) and a microplate reader (Labsystems Multiskan MS 352; Thermo Fisher Scientific, Waltham, MA, USA), with a sensitivity >84% and specificity >98%. All samples were prepared in duplicate and average values were used for analysis. Intra- and inter-assay coefficients of variation were <9% and <11%, respectively.
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