The procedures used to obtain and characterize AGEs-Csn as well as to quantify the AGEs in AGEs-Csn were previously described69 (link). Briefly, 28 mg/mL total Csn was dispersed in 30 mM phosphate buffer saline (PBS) (pH 6.8) in the presence of 116 mM lactose and 55 mM glucose-fructose and was subjected to heat treatment that simulated the steps used in the production of flavoured ultra-high-temperature processing (UHT) milk (70 °C for 30 min and 135 °C for 8 seconds). Subsequently, to simulate the prolonged storage of UHT products, the samples were incubated at 25 °C for 90 days, and the unreacted reducing sugars were removed by filtration using 10-kDa cut-off membrane filter units (Millipore, St. Charles, MO, USA). The total AGEs content of AGEs-Csn and of the control (non-glycated Csn) was 267 and 11 µg/mg protein69 (link), respectively. Any bacterial endotoxin contamination was removed from the non-glycated Csn and AGEs-Csn solutions using an Endotoxin Removal Standard Kit (Bio-Rad, Hercules, CA, USA). The success of the purification procedure was confirmed by measuring endotoxin levels in AGEs-Csn and non-glycated Csn solutions using an E-toxate kit (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were determined using a Bradford-based assay (Bio-Rad) with BSA as a standard.
Bradford based assay
Manufactured by Bio-Rad
Sourced in Italy
The Bradford-based assay is a colorimetric assay used for the quantitative determination of protein concentration. It relies on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
E toxate kit, by Merck Group (1 mentions)
A12214, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
Il 13, by Miltenyi Biotec (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
α tubulin, by Merck Group (1 mentions)
Hybond ecl membrane, by Cytiva (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Clone hecd1, by Thermo Fisher Scientific (1 mentions)
Hybond, by Cytiva (1 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
9 protocols using bradford based assay
1
Detailed Protocol for AGEs-Casein Preparation
2
Cytokine Release Quantification
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The release of IL-6, TNF-α, IL-10 and CXCL8 in the culture supernatant was measured in duplicate determinations by commercially available ELISA kits (R&D, Minneapolis, MN USA) according to the manufacturer's instructions. Since the number of adherent monocytes can vary in each well and in different experiments, the results were normalized for the total protein content in each well, determined in the cell lysates (0.1% Triton X-100) by a Bradford based assay (Biorad).
3
Fluorometric Assay for MAOB Activity
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MAOB activity in nigrostriatal tissues of MAOB KO, WT, and GFAP-MAOB mice was determined using a commercially available kit (A12214; Thermo Fisher Scientific), following the manufacturer’s protocol. Lysates were prepared from each region of the transgenic mouse brain by centrifuging at 16,000 rpm after homogenization with RIPA lysis buffer with phosphatase and protease inhibitor cocktail (Roche, Switzerland). Protein concentration in the supernatant was determined using a Bradford-based assay (Bio-Rad Laboratories, Hercules, CA, USA), and samples were diluted to 500 mg per 2 ml total volume with reaction buffer. Samples were pre-incubated for 30 min at room temperature with the specific MAOB inhibitor, pargyline hydrochloride (1 μM). After incubation, samples were added to individual wells of a 96-well microplate. The fluorometric assay was initiated by adding 100 μl of a reaction mixture containing Amplex Red reagent (400 μM), horseradish peroxidase (HRP; 2 U/ml), and benzylamine (2 mM), a specific substrate of MAOB. Plates were incubated for 30 min at room temperature, protected from light, and fluorescence was measured at excitation and emission wavelengths of 550 nm and 590 nm, respectively, using a microplate fluorometer (Life Sciences, Manchester, UK). H2O2 (10 μM) was used as a positive control, and reaction buffer alone was used as a negative control.
4
Cytokine Stimulation of Immune Cells
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HLMs, monocytes, and MDMs were cultured in 24-well plates in RPMI 1640 supplemented with 5% FBS (Sigma-Aldrich, Milan, Italy), 2 mM l-glutamine, and 1% antibiotic-antimycotic solution.
The cells were treated with IL-13 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), IL-4 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), detoxified LPS (100 ng/mL) (from Escherichia coli serotype 0111:B4; Sigma-Aldrich, Milan, Italy), or TSLP (5 ng/mL) for 16 h or 6 h at 37 °C. In selected experiments, the cells were preincubated (30 min, 37 °C) with or without actinomycin D (1 μg/mL) and then stimulated (16 h, 37 °C) with LPS or IL-4. At the end of incubation, the supernatants were collected and stored at −80 °C for subsequent ELISA quantification of cytokines. Lysis of the cells in the plates was carried out by using 0.1% Triton X-100 for total protein quantification by a Bradford-based assay (Bio-Rad, Segrate, MI, Italy).
The cells were treated with IL-13 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), IL-4 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), detoxified LPS (100 ng/mL) (from Escherichia coli serotype 0111:B4; Sigma-Aldrich, Milan, Italy), or TSLP (5 ng/mL) for 16 h or 6 h at 37 °C. In selected experiments, the cells were preincubated (30 min, 37 °C) with or without actinomycin D (1 μg/mL) and then stimulated (16 h, 37 °C) with LPS or IL-4. At the end of incubation, the supernatants were collected and stored at −80 °C for subsequent ELISA quantification of cytokines. Lysis of the cells in the plates was carried out by using 0.1% Triton X-100 for total protein quantification by a Bradford-based assay (Bio-Rad, Segrate, MI, Italy).
5
Isoprene Oxidation Kinetics Assay
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Isoprene oxidation was monitored by measuring the concentration of isoprene in the headspace using a Fast Isoprene Sensor (Hills Scientific) (56 (link)). Assays were performed using 25-mL vials containing 1 mL of Rhodococcus sp. AD45 cell extract (4.5 to 5.5 mg mL−1 protein) and 0.5 mL of 50 mM phosphate buffer (pH 7.0) and/or purified protein components at a final concentration of 5 μM, in the presence of 5 mM DTT. Cell extract was prepared by the resuspension of frozen cell pellets in 50 mM phosphate buffer (pH 7.0) and 5 mM DTT. The cells were broken by three passes through a French pressure cell at 20,000 lb/in2 (137 MPa). Unbroken cells and cell debris were removed by centrifugation at 16,000 × g (4°C, 30 min). Protein concentration was determined using a Bradford-based assay (Bio-Rad). Once sealed with butyl stoppers, ∼200 ppmv isoprene was added to the headspace, vials were transferred to a 30°C water bath with orbital shaking and left to equilibrate for 3 min. The reaction was initiated by the addition of 5 mM NADH, and 50-μL headspace samples were analyzed every 3 min for a total of 15 min using a Fast Isoprene Sensor (Hills Scientific). The data were analyzed using the QtiPlot program (version 5.6.1, Ion Vasilief), and compared to standards ranging between 70 and 500 ppmv isoprene to determine the rate of isoprene oxidation.
6
Protein Extraction and Western Blot Analysis
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Protein lysates were prepared using catenin lysis buffer [1% Triton X-100 (Sigma) and 1% IGEPAL CA-630 (Sigma) in PBS] supplemented with protease and phosphatase inhibitor cocktails (Roche and Sigma, respectively). Protein concentration was determined using a Bradford-based assay (Bio-Rad). For analysis of total protein samples, 20-40 µg of protein were eluted in sample buffer, separated in 10% or 15% SDS–polyacrylamide gels (SDS-PAGE), and electroblotted onto Hybond ECL membranes (Amersham Biosciences). Membranes were blocked with 5% non-fat milk or 5% BSA in 0.5% Tween-20 in PBS for 1 h, and immunoblotted with antibodies against E-cadherin (1:1000, Clone HECD1, Invitrogen), REG1A (1:500, Invitrogen) and α-Tubulin (1:10,000, Sigma). The secondary antibodies sheep anti-mouse or donkey anti-rabbit HRP-conjugated (Amersham Biosciences) were then incubated, followed by detection with ECL reagents (Bio-Rad). Protein bands were quantified using the Quantity One 4.6.9 Software (Bio-Rad).
7
Extraction and Detection of Nuclear Proteins
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Nuclear Extracts were prepared with NucBusterTM Protein Extraction Kit (Novagen) and measured by Bradford-based assay (BioRad). The extracts were stored at −80°C and thawed on ice with the addition of protease inhibitor Cocktail (Roche) directly before use. 15 μg of nuclear extracts were run on 10% acrylamide gels and transferred to hydrophobic polyvinylidene difluoride (PVDF) membrane (GE-Amersham, 0.45 μm) using BioRad Semi-dry system, then visualized by exposure to MyECL Imager (Thermo Scientific).
Rabbit polyclonal antibodies were generated by injection of synthetic peptides corresponding to the tether regions of ZSCAN5A (DLVRAKEGKDPPKIAS) and mouse Zscan5b proteins (CPEPANPQPEKQVDSL); peptide synthesis, antibody production, and affinity purification of antibodies against the purified peptide epitope we carried out by Abgent Inc. ZSCAN5B (sc-249845, Santa Cruz Biotechnology) and ZSCAN5D (ARP47809_P050, Aviva Systems Biology) antibodies were obtained from commercial sources. Antibody preparations were tested for protein specificity and efficiency by Western blot staining along with anti-TATA binding protein control antibody (1TBP18, Abcam),
Rabbit polyclonal antibodies were generated by injection of synthetic peptides corresponding to the tether regions of ZSCAN5A (DLVRAKEGKDPPKIAS) and mouse Zscan5b proteins (CPEPANPQPEKQVDSL); peptide synthesis, antibody production, and affinity purification of antibodies against the purified peptide epitope we carried out by Abgent Inc. ZSCAN5B (sc-249845, Santa Cruz Biotechnology) and ZSCAN5D (ARP47809_P050, Aviva Systems Biology) antibodies were obtained from commercial sources. Antibody preparations were tested for protein specificity and efficiency by Western blot staining along with anti-TATA binding protein control antibody (1TBP18, Abcam),
8
Protein Concentration Determination
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At the end of each treatment interval, culture supernatants were harvested and centrifuged to remove cell debris and were then concentrated using a 3-kDa cut-off membrane filter unit (Millipore). The protein concentration was determined using a Bradford-based assay (Bio-Rad) with BSA as a standard71 (link). The conditioned media samples were aliquoted and stored at −80 °C.
9
TSLP Regulation of VEGF-A Production
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HLMs were cultured in 24-well plates in RPMI 1640 medium supplemented with 5% FCS, 2 mM l-glutamine, and 1% antibiotic–antimycotic solution, as previously described [17 (link)]. HLMs were treated with TSLP and its proteolytic fragments for 16 h at 37 °C. At the end of incubations, the supernatants were collected and stored at −80 °C for subsequent ELISA quantification of VEGF-A. Cell lysis in the plates was carried out using 0.1% Triton X-100 for total protein quantification by a Bradford-based assay (Bio-Rad, Segrate, Milan, Italy).
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