HUVECs seeded at a density of 104/cm2 in cell culture Petri dishes (TPP) were exposed for 24 h to two doses of each T. marschallianus extract—TMCE: 0.94 μg GAE/mL (sample TMCE1) and 0.094 μg GAE/mL (sample TMCE2) and TMSE: 0.66 μg GAE/mL (sample TMSE1) and 0.066 μg GAE/mL (sample TMSE2)—either in medium with 200 mmol/L glucose (hyperglycemic stress) or in medium with 137 mmol/L glucose (normoglycemic conditions). Then, the cells were washed three times and incubated for another 24 h in standard conditions. After that, the cells were collected by scraping and treated with a lyses buffer containing IGEPAL Nonidet 1% (Sigma-Aldrich, Darmstadt, Germany) and 1% protease inhibitor complex (Sigma) in PBS for 1 h on ice. The protein content was determined by the Bradford method (Bio-Rad, Hercules, California, USA) according to the manufacturer’s specifications. All the experiments were performed in triplicate. For all assays the lysates were corrected by total protein concentration. The cell lysates were used for MDA assessment and Western blot analysis.
Protease inhibitor complex
Manufactured by Merck Group
Sourced in United States
Protease inhibitor complex is a type of laboratory equipment used to inhibit the activity of proteases, which are enzymes that break down proteins. The core function of this product is to provide a reliable and effective way to control and study proteolytic processes in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Bradford method, by Bio-Rad (3 mentions)
Igepal nonidet, by Merck Group (2 mentions)
Anti p38β, by Santa Cruz Biotechnology (1 mentions)
Gentamicin, by Harvard Bioscience (1 mentions)
Amphotericin, by Harvard Bioscience (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Human umbilical vein endothelial cells (huvec), by European Collection of Cell Cultures (1 mentions)
Doxycycline hyclate, by Merck Group (1 mentions)
Esgro, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Laemli sample buffer, by Bio-Rad (1 mentions)
Catch and release v2.0 reversible immunoprecipitation system, by Merck Group (1 mentions)
8 protocols using protease inhibitor complex
1
HUVEC Exposure to T. marschallianus Extracts
2
Interaction Between p38β and MnSOD
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Neonatal rat cardiomyocytes were synchronized overnight in serum-free medium, and H/R was applied as described above. Cells were then lysed in lysis buffer (50 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 40 mM β-GP, 200 µM Na3VO4, 40 mM p-NPP, 1 mM PMSF, protease inhibitor complex, and phosphatase inhibitor complex [Sigma-Aldrich]). p38β or MnSOD from the lysate was then immunoprecipitated overnight at 4°C using polyclonal anti-p38β (Santa Cruz Biotechnology, sc-6187-R) or anti-MnSOD antibody (Santa Cruz Biotechnology, sc-30080), respectively, conjugated to Sepharose beads. The immunoprecipitated protein complex was loaded onto 12% SDS-PAGE gel, electrophoresed and processed for immunoblotting. To determine a molecular interaction between p38β and MnSOD, the p38β-immunoprecipitating complex was blotted for MnSOD and vice versa.
3
Endothelial Cell Responses to Phytochemicals
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Commercial human umbilical vein endothelial cells (HUVEC) were bought from the European Collection of Cell Cultures (ECACC, Porton Down, Salisbury, UK) and multiplied in RPMI medium, supplemented with 10% fetal calf serum (FCS), gentamicin 50 μg/ml, and amphotericin 100 μg/ml (Biochrom AG, Berlin, Germany) in a humidified CO2 incubator at 37°C. Cell cultures in the 23rd to 26th passages were used. The analysis of surface markers was performed by flow cytometry (BD FACS Canto II flow cytometer, Becton Dickinson & Company, Franklin Lakes, NJ, USA) and used different monoclonal antibodies (ICAM-1, CD29, CD34, CD73, CD90, and CD105).
Cells seeded at a density of 104/cm2 in cell culture Petri dishes (TPP) were settled for 24 h in medium then exposed for 24 h to either Equisetum arvense L. extract (25 or 2.5 μg/ml), caffeic acid (10 or 1 μg/ml), and cathechin (10 or 1 μg/ml), as positive controls; afterwards, the cells were washed 3 times with PBS and incubated for 24 h either in normotonic (137 mmol/l) or hypertonic conditions (200 mmol/l). Cells were collected by scraping and treated with a lysis buffer containing IGEPAL-Nonidet 1% (Sigma) and 1% protease inhibitor complex (Sigma) in PBS for 1 h, on ice. The protein content was determined by the Bradford method (Bio-Rad, USA). All the experiments were performed in triplicate.
Cells seeded at a density of 104/cm2 in cell culture Petri dishes (TPP) were settled for 24 h in medium then exposed for 24 h to either Equisetum arvense L. extract (25 or 2.5 μg/ml), caffeic acid (10 or 1 μg/ml), and cathechin (10 or 1 μg/ml), as positive controls; afterwards, the cells were washed 3 times with PBS and incubated for 24 h either in normotonic (137 mmol/l) or hypertonic conditions (200 mmol/l). Cells were collected by scraping and treated with a lysis buffer containing IGEPAL-Nonidet 1% (Sigma) and 1% protease inhibitor complex (Sigma) in PBS for 1 h, on ice. The protein content was determined by the Bradford method (Bio-Rad, USA). All the experiments were performed in triplicate.
4
ChIP-seq of HA and MacroH2A2
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ES cells were grown in standard ES media containing Lif (ES Gro, Millipore) on mitotically inactivated feeder MEFs until approximately 80% confluence. ES cells were then pre-plated on gelatin and incubated for 45 min to deplete feeder MEFs by virtue of their faster adherence than ES cells (roughly 3 hours). ES cells were then split onto three gelatinized plates each of which was induced at different time points by the addition of final 2 µg/mL doxycycline hyclate (Sigma). A similar procedure was used for induction of MEFs at passage 2. All time points were crosslinked with formaldehyde to a final concentration of 1% for 10 minutes, and were quenched with 125 mM glycine. Crosslinked cells were resuspended in 270 µl SDS-Lysis Buffer (1% SDS, 10 mM EDTA and 50 mM Tris-Cl, pH 8.1) including protease inhibitor complex (Sigma) and PMSF (Sigma), and chromatin was sonicated in Bioruptor (UCD-200) to an average size of 150–400 base pairs. 70 µg of chromatin of each time point was immunoprecipitated either with HA antibody (Abcam) or MacroH2A2 antibody (Abcam). Eluted ChIP materials were PCI (Phenol-Chloroform-Isoamylalcohol) extracted, RNAse (Qiagen) and CIP (NEB) treated.
5
TGF-β Induced Hepatic Stellate Cell Modulation
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LX-2 cells were plated at 5 × 104 cells/mL in 24-well plates and were cultured in growth medium supplemented with FBS 1%. Half of the cells were incubated with 10 ng/mL TGF-β for 24 h. Afterwards, the cells were treated with VL, NPCS and NPCS-VL. The cells were divided into eight groups as follows: I: untreated cells (negative control group); II: received only 10 ng/mL TGF-β; III: received only 25 μg/mL NPCS; IV: received 10 ng/mL TGF-β + 25 μg/mL NPCS; V: received only 25 μg/mL VL; VI: received 10 ng/mL TGF-β + 25 μg/mL VL; VII: received 25 μg/mL NPCS + 25 μg/mL VL; VIII: received 10 ng/mL TGF-β + 25 μg/mL NPCS + 25 μg/mL VL. All tests were performed in triplicate. The cell lysates used for the biochemical assays were obtained using the following method: the cells were collected from the surface of the culture, placed in ice, rinsed twice in the buffer solution and then lysed in 0.3 mL cold solution of 0.1% Triton X-100, 0.25 mol/L NaCl, 1 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L dithiothreitol, 0.1 mmol/L Na3VO4, 50 mmol/L TRIS HCl and protease-inhibitor complex 1% (Sigma). Lysates were centrifuged and protein concentration in cell culture supernatant was determined using the Bradford method, according to the manufacturer’s specifications (Bio-Rad Laboratories, Richmond, CA, USA).
6
Protein Extraction from Cells and EVs
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Total protein was extracted from LN18 and LN229 cells, or isolated EV pellets, in the presence of RIPA+ buffer (Sigma-Aldrich, Saint Louis, MO 63103, USA) containing 10% protease inhibitor complex (Sigma-Aldrich), pipetting gently with regular intervals while shaking the cell preparation on ice for 2 h. Thereafter the cell or EV preparations were centrifuged at 16,000× g (4 °C/20 min) and the supernatant containing the extracted protein collected. Protein extracts were either used immediately for immunoprecipitation and proteomic analysis, or re-constituted in 2× Laemmli sample buffer for Western blotting.
7
Tissue Homogenization and Protein Extraction
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Muscle, liver and POAT preparation for WB Samples of snap-frozen tissue samples were homogenized in lysis buffer containing Igepal-nonidet 1% (Sigma-Aldrich Chemicals GmbH), 1% protease inhibitor complex (Sigma-Aldrich Chemicals GmbH) in PBS for 1 h, on ice. Cell extracts were spun at 14 000 g for 30 min at 4 8C. Supernatant was collected and 50 ml were used to determine the protein content by the Bradford method. Lysates were mixed 1:2 (v/v) with Laemli sample buffer (Bio-Rad) containing 2-mercaptoethanol and the proteins were denaturized at 95 8C for 10 min. The protein concentration in each sample was 4 mg/ml.
8
Protein Extraction from Cod Mucosa EVs
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For extraction of protein, EV pellets derived from cod mucosa were resuspended in RIPA+ buffer (Radioimmunoprecipitation assay buffer containing 10% protease inhibitor complex; Sigma-Aldrich, U.S.A), pipetting gently at regular intervals for 2 h on ice. Protein was isolated by centrifugation at 16,000 g for 20 min and collecting the supernatant. For isolation of total deiminated proteins from the EV protein preparation, immunoprecipitation was performed using the Catch and Release® v2.0 Reversible Immunoprecipitation System (Merck, U.K.) according to the manufacturer's instructions in conjunction with the monoclonal F95 pan-deimination antibody (MABN328 Merck, U.K.), which is raised against a deca-citrullinated peptide and specifically detects protein citrulline (Nicholas and Whitaker, 2002) . Incubation was performed overnight at 4 °C on a rotating platform. F95 bound proteins were thereafter eluted under reducing conditions according to the manufacturer's instructions (Merck) and the F95 enriched eluate was analysed by liquid chromatography-mass spectrometry (LC-MS/MS; performed by Cambridge Centre for Proteomics, U.K.) with peak list files submitted to in-house Mascot (Cambridge Centre for Proteomics), using the following database: Gadus_morhua_20190405 (1283 sequences; 308668 residues), and with setting set at significance threshold p<0.05 and cut-off at Ions score 20.
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