Samples of the periarticular tissues over the TMJ were dissected as described by Lamana and collaborators (2017) (link). Briefly, the standard sample size was 1 × 1 × 0.5 cm, which includes temporalis, masseter, and pterygoideus externus muscles, articular cartilage, disc fibrocartilage, and ligaments. Samples were stored at −80°C until processing. Tissues samples were homogenized in 500 μl of the appropriate buffer containing protease inhibitors (Ripa Lyses Buffer, Santa Cruz, Biotechnology, Dallas, Texas, USA) using a specific sample homogenizer (BeadBlaster™ 24, Benchmark, Triple-Pure High Impact Zirconium Beads, Beads, Ø: 1.0mm). After 4 cycles of 30 seconds with resting period of 40 seconds, the samples were centrifuged for 10 min/10.000 rpm or 12298 RCF/ 4°C. The total amount of extracted proteins was colorimetrically measured using the micro bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL, USA). The supernatants were stored at −20°C until further analysis.
Micro bicinchoninic acid bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantitation method used to measure the total protein concentration in a sample. The kit utilizes bicinchoninic acid as the detection reagent, which reacts with the peptide bonds in proteins to produce a purple-colored complex that can be measured spectrophotometrically.
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Lab products found in correlation
Iodoacetamide, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Milli q system, by Merck Group (2 mentions)
Steritop, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Amplex red cholesterol assay kit, by Thermo Fisher Scientific (1 mentions)
Cell scraper, by Techno Plastic Products (1 mentions)
Sebacic acid, by Merck Group (1 mentions)
Glutaraldehyde solution, by Merck Group (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Merck Group (1 mentions)
1 1 1 3 3 3 hexafluoro 2 propanol hfip, by ABCR (1 mentions)
Amylase activity assay kit, by Merck Group (1 mentions)
4 octanol, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Gallic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Folin ciocalteu reagent, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Vanillin, by Merck Group (1 mentions)
16 protocols using micro bicinchoninic acid bca protein assay kit
1
Periarticular TMJ Tissue Dissection
2
Exosome Isolation from THP-1 Cell Conditioned Media
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80% confluent THP-1 cell lines cultured for an additional 48 hours in DMEM media deprived of FBS. The conditioned media of THP-1 cell lines was obtained and centrifuged at 300 × g for 10 min and 2000 × g for 10 min at 4°C to remove dead cells and cellular debris. Subsequently, the supernatant was filtered using a 0.45 μm filter sterilize Steritop TM (Millipore) to remove residual dead cells and cellular debris. Hereafter, the supernatant was centrifuged at 4000 × g at 4°C to about 200 μL by ultra-filtration in a 15 mL Amicon Ultra-15 Centrifugal Filter Unit (Millipore) and then centrifuged at 100,000 × g for one hour at 4°C to pellet the small vesicles. Exosomes were stored at − 80°C or used for downstream experiments. The protein concentration of the exosomes was determined using the Micro Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher, Waltham, MA, USA).
3
Cellular Cholesterol Quantification
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Cultured cells were washed two times with PBS to remove any residual media and lysed with 200 μl of 0.1% Triton X-100 for total cellular cholesterol quantification using the Amplex red cholesterol assay kit the per manufacturer's instructions (Thermo Fisher). Total cholesterol content was normalized to total cellular protein quantified with the micro-bicinchoninic acid (BCA) protein assay kit per the manufacturer's instructions (Thermo Fisher). Briefly, total cholesterol (in micrograms) was divided by total protein (in milligrams) for each sample.
4
Protein Extraction from IEC-6 Cells
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IEC-6 cells were seeded onto 6-well plates at 5 × 105 cells per well in 3 mL of DMEM containing 10% FBS, 4 µg/mL insulin, 100 U/mL penicillin, and 100 µg/mL streptomycin. After 3 days of culture, HK L-137 (500 μg/mL) was added. After 24 h, the supernatant was removed, and the wells were washed with ice-cold PBS. After removal of PBS, ice-cold RIPA buffer supplemented with protease inhibitor cocktail tablets and phosphatase inhibitor tablets was added, and the cells were collected with a cell scraper (Techno Plastic Products AG, Trasadingen, Switzerland). After 1 h on ice, the extract solution was centrifuged at 16,260g for 30 min at 4 °C, and the supernatant was collected. Total protein levels in the preparations were determined with a Micro Bicinchoninic Acid (BCA) Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA).
5
Fabrication of Poly(Pyrrole) Hydrogels
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PPy, sebacic acid, and glutaraldehyde solution (25 wt % in H2O) were purchased from Sigma-Aldrich. Glycerol 85% was purchased
from Yliopiston Apteekki, Finland. Sodium dodecyl sulfate (SDS), dimethylsulfoxide
(DMSO), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
were purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS)
10×, FBS, Dulbecco’s modified Eagle medium (DMEM),l -glutamine, nonessential amino acids (NEAAs), and penicillin–streptomycin
were purchased from HyClone. Hank’s buffered salt solution
(HBSS) 10× was obtained from Gibco Life Technologies, and micro-bicinchoninic
acid (BCA) protein assay kit was obtained from Thermo Fisher Scientific.
The small molecule compound 3i-1000 was purchased from Pharmatory
Ltd. (Oulu, Finland). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was
purchased from abcr, Germany, and Collagen Type I, Calf Skin Lyophilized
was obtained from EPC.
from Yliopiston Apteekki, Finland. Sodium dodecyl sulfate (SDS), dimethylsulfoxide
(DMSO), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
were purchased from Sigma-Aldrich. Phosphate-buffered saline (PBS)
10×, FBS, Dulbecco’s modified Eagle medium (DMEM),
were purchased from HyClone. Hank’s buffered salt solution
(HBSS) 10× was obtained from Gibco Life Technologies, and micro-bicinchoninic
acid (BCA) protein assay kit was obtained from Thermo Fisher Scientific.
The small molecule compound 3i-1000 was purchased from Pharmatory
Ltd. (Oulu, Finland). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was
purchased from abcr, Germany, and Collagen Type I, Calf Skin Lyophilized
was obtained from EPC.
6
Analytical Protocol for Phenolic Profiling in Shiraz Wines
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All solvents and chemicals used were of analytical grade. Methanol, Folin-Ciocalteu reagent, concentrated hydrochloric acid sodium carbonate, gallic acid, vanillin, catechin, 1 M triethylammonium bicarbonate (TEAB) buffer, sodium chloride, acetone, urea, iodoacetamide, tris(2-carboxyethyl)phosphine hydrochloride (TECP), acetonitrile (ACN), trifluoroacetic acid (TFA), β-lactoglobulin, phosphate-buffered saline, 4-octanol used in the GC analysis and amylase activity assay kit were purchased from Sigma-Aldrich (Castle Hill NSW, Australia). Micro bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). Purified water was obtained from a Milli-Q system (Millipore Australia, Bayswater, Victoria, Australia). Eight Shiraz wines produced in Victoria, Australia, were purchased from local wine producers. Due to the same wine samples being no longer available on the market when the spiking tests were conducted, Wine 8 of vintage 2019 was used for the spiking tests.
7
Characterization of Hyaluronic Acid Hydrogels
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Unless stated otherwise, chemicals were purchased from Sigma Aldrich (Schnelldorf, Germany) and used as received. Sodium HA was purchased from Lifecore, Biomedical (Chaska, MN, USA). A polyethylene oxide/polyethylene glycol calibration kit for gel permeation chromatography (GPC) was purchased from Agilent Technologies (Santa Clara, CA, USA). A micro bicinchoninic acid (BCA) protein assay kit and lactate dehydrogenase (LDH) cytotoxicity assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fluorescent stains 4′,6-diamidino-2-phenylindole (DAPI) and LysoTrackerTM Deep Red were purchased from Vector Laboratories (Burlingame, CA, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Additionally, 4-(dimethylamino) pyridinium-4-toluenesulfonate (DPTS) was previously synthesized in laboratory according to the procedure described by Moore et al. [62 (link)]. Ultrapure water was produced in the laboratory using a Milli-Q® system (Merck Millipore, Darmstadt, Germany) with a resistivity at 18.2 MΩ-cm.
8
Protein Extraction and Quantification
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Amicon Ultra-0.5 centrifugal filters (cut-off 3 kDa) were purchased from Millipore (Bedford, MA, USA). The micro bicinchoninic acid (BCA) protein assay kit was from Thermo Scientific (Rockford, IL, USA). Trypsin (sequencing grade) was purchased from Promega Corporation (Madison, WI, USA). Calcium chloride, formic acid (FA), hydrochloric acid, and ammonium acetate were purchased from Merck (Darmstadt, Germany). Dithiothreitol (DTT) and iodoacetamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN) was purchased from Lab-scan (Dublin, Ireland).
9
Protein Extraction and Quantification Protocol for Western Blotting of hVICs
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For Western blotting analysis, hVICs from each treatment were harvested after 3 times wash with PBS. Samples were homogenized with a Dounce grinder with a tight pestle in ice-cold lysis buffer (15 mM HEPES, pH 7.2, 60 mM KCl, 10 mM NaCl, 15 mM MgCl2, 250 mM Sucrose, 1 mM EGTA, 5 mM EDTA, 1 mM PMSF, 2 mM NaF, 4 mM Na3VO4). A protease inhibitor cocktail (Sigma–Aldrich) was included in the isolation buffer to prevent protein degradation. The lysate was stored at −80 °C for later use. The concentration of the total protein extracted was estimated by Micro Bicinchoninic acid (BCA) Protein Assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
10
Tissue Fractionation and Protein Quantification
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ICR mice were purchased from Japan SLC (Shizuoka, Japan) and raised in our laboratory. Cytosol and membrane extracts of various tissues were prepared from postnatal day 40 (P40) mice of either sex as described previously [7 (link)]. Briefly, tissues were homogenized with 10 vol. of 50 mM Tris/pH 7.5 containing 0.1 M NaF, 5 mM EDTA, 1 mM Na3VO4, 10 μg/ml aprotinin and 10 μg/ml leupeptin (buffer S). Each suspension was sonicated at 0°C for 1 min and centrifuged at 125,000 g for 20 min at 4°C. The supernatants were used as cytosolic extracts and the pellets were washed once by sonication and centrifugation with buffer S and then solubilized with buffer S containing 2% SDS. In some experiments, brain tissues were directly homogenized with buffer S containing 2% SDS to obtain the whole tissue extracts [6 (link)]. Protein concentration was estimated with a micro bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin (BSA) as a standard. Western blotting was done as described earlier.
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