Tissue preparation was adapted from our previously published methods.14 (link)–17 Tissue samples (11.6–22.5 mg wet weight) were homogenised with a hand-held dounce (Omni BioMasher, Georgia, USA) in 3 × tissue weight of Tris buffered saline (TBS: 50 mM Tris; 150 mM NaCl; pH 8.0) containing EDTA free protease inhibitors (Roche, NSW, Australia). Samples were centrifuged at 16,000 g for 15 min at 4°C before the supernatant fraction was collected and stored at −80°C. This was termed the ‘soluble fraction’, and represented all cytosolic proteins and the interstitium. The remaining tissue pellet was resuspended in 3 × tissue weight of urea buffer (7M urea; 2M thiourea, 4% 3-[(3-cholamindopropyl) dimethylammonio]-1-propanesulfonate (CHAPS); 30mM Bicine; pH 8.5; Sigma, NSW, Australia) and centrifuged 16,000 g for 30 min at 4°C and the resultant supernatant was collected and stored at −80°C. This was termed the ‘membrane fraction’, representing both membrane-bound proteins and those encased within cellular organelles. The final pellet was resuspended in 3 × tissue weight of 70% formic acid overnight before being centrifuged at 16,000 g for 30 min and the resultant supernatant collected and stored at −80°C. This was termed the ‘insoluble fraction’, and represented all material not previously extracted. No observable material remained following the final preparation step.
Bicine
Manufactured by Merck Group
Sourced in United States
Bicine is a chemical compound used in various laboratory applications. It functions as a buffering agent, maintaining a stable pH environment in chemical and biological experiments.
Automatically generated - may contain errors
Lab products found in correlation
Resazurin, by Merck Group (3 mentions)
Alcohol dehydrogenase, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Silver nitrate, by Merck Group (2 mentions)
Sodium borohydride, by Merck Group (2 mentions)
Dopamine hydrochloride, by Merck Group (2 mentions)
Hydroquinone, by Merck Group (2 mentions)
Gold 3 chloride hydrate, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Cd38 antibody, by Santa Cruz Biotechnology (2 mentions)
Diaphorase, by Merck Group (2 mentions)
8 br cadpr, by Merck Group (2 mentions)
Adp ribosyl cyclase, by Merck Group (2 mentions)
Phosphodiesterase 1, by Worthington (2 mentions)
Edta free protease inhibitor, by Roche (1 mentions)
Cetyltrimethylammonium bromide, by Merck Group (1 mentions)
Copper 2 chloride dihydrate, by Merck Group (1 mentions)
Tem grid, by Ted Pella (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
18 protocols using bicine
1
Protein Fractionation from Tissue
2
Synthesis of Multimodal Nanoparticles
3
Synthesis of Gold Nanoparticles with CTAB
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Gold(III) chloride hydrate (HAuCl4), cetyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4), hydroquinone, silver nitrate (AgNO3), dopamine hydrochloride, bicine, resazurin, and McCoy's 5A medium were purchased from Sigma-Aldrich (Atlanta, GA, USA). Methoxy poly(ethylene glycol) thiol (HS-mPEG) with molecular weights (Mw) of 2k, 5k, 10k, and 20k were purchased from Nanocs. Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Fisher. Calcein AM (4 mM) dissolved in DMSO was purchased from Biotium. Propidium iodide (≥ 95%, PI) was purchased from Combi-Blocks (San Diego, CA, USA). All reagents were used without further purification. Deionized water (18.2 MΩ·cm) purified with a Milli-Q Academic water purification system was used to make aqueous solutions.
4
Colorimetric Assay for Antioxidant Capacity
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Glucose oxidase (GOx) from Aspergillus niger (Type X-S, 149 500 units per g solid), polyethyleneimine (PEI, branched, average Mw 25 000 by light scattering), pyrogallol (PG), bicine, gallic acid, and the Folin–Ciocalteu reagent (10 N) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Catechol (CT) and phosphate-buffered saline (PBS, 10× solution) were purchased from Fisher (Waltham, MA, USA). Glucose, horseradish peroxidase (HRP, RZ 3.0), and 3,3′,5,5′-tetramethylbenzidine (TMB) were procured from Santa Cruz Biotechnology (Dallas, TX, USA). Urea was obtained from Panreac Quimica (Barcelona, Spain). Ultrapure water was prepared using a 5-UV water purification system (EMD Millipore).
5
Aβ40 Peptide Synthesis and Purification
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Aβ40, H2N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV -COOH, was synthesized and purified using a stepwise solid phase peptide synthesis protocol as previously reported [15 (link)]. Bicine, PBS, 1,1,1,3,3,3-hexafluoro-2-propanesulfonic acid (HFIP), anhydrous dimethylsulfoxide (DMSO), Tris(2,2’bipyridyl)dichlororuthenium(II) (Ru(Bpy)) and ammonium persulfate (APS) were obtained from Sigma-Aldrich. SDS-PAGE apparatus, 15% SDS-PAGE gel, and 10X Tris-glycine SDS Buffer were purchased from Bio-Rad. PBS (1X) consists of phosphate buffer concentration of 0.01M and a sodium chloride concentration of 0.154M. Lane marker reducing sample buffer (5X) was from Thermo Scientific and protein marker was purchased GE Healthcare. Silver staining kit was acquired from Amersham Biosciences. All other reagents, solvents, and chemicals were of the highest purity commercially available and used as received.
6
Biomimetic PCL-DPP-PCL Scaffold Functionalization
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PCL-DPP-PCL scaffolds were pre-wet with 70% ethanol followed by DI water. Thereafter, they were incubated in 0.5 mg/mL of 3,4-dihydroxy-L-phenylalanine (DOPA, Sigma), which was dissolved in poly-DOPA coating buffer that comprised of 10 mM Bicine (Sigma) and 250 mM NaCl (pH 8.5, Sigma) for 4 hours at 150 r/min. The scaffolds were then washed with DI water three times, 30 min/time and lyophilized overnight. Subsequently, they were incubated in poly-D-lysine solution (PDL, 5.0 μg/mL, Sigma) at 37°C and 100 r/min overnight. On the next day, the scaffolds were washed twice with PBS (10010-023, Thermo Fisher Scientific) for 15 minutes each time and then immersed in Dulbecco’s modified Eagle’s medium (DMEM, 11960-044, Thermo Fisher Scientific) until cell seeding.
7
Synthesis and Characterization of Polyserotonin Nanoparticles
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Polyserotonin nanoparticles (PSeNP) were synthesized using four different buffers: (1) trisaminomethane (Tris), (2) 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid (HEPES), (3) diethanolamine (DEA), and (4) bis (2-hydroxylethyl)amino acetic acid (Bicine), all purchased from Sigma-Aldrich (Oakville, ON, Canada) (see Scheme 1 for chemical structures). Briefly, 100 mL of 10 mM buffer at pH 9 was placed in a 500 mL single-neck round-bottom flask and heated in a water bath to 60 °C. Serotonin hydrochloride (2 mg/mL) (Tokyo Chemical Industry America, Tokyo, Japan) was then added, stirred, and capped for 45 min. Subsequently, the mixture was purified by centrifugation (Eppendorf centrifuge 5804R) at 16,639 rcf at 4 °C for 5 min. Pellets were removed, while allowing the supernatant to further grow in the dark for an additional 24 h. The nanoparticles were then centrifuged out at 16,639 rcf at 4 °C for 1 h followed by triple washings with milli-Q water at 20,817 rcf at 4 °C for 15 min. In addition to nanoparticles, PSe films were also prepared by incubating coverslips in the supernatant for 24 h.
8
Iron Speciation Analysis Procedure
9
Oleic Acid Vesicles and Micelles Protocol
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Fatty acid
vesicles were prepared
by mixing 10 mM liquid oleic acid (Nu-Check, Elysian, MN), 20 nM biotin-PEG(5K)-DSPE
(biotin–poly(ethylene glycol)–distearoylglycerophosphoethanolamine,
Nanocs, Boston, MA, 100 μM stock in water), 10 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonate,
Sigma-Aldrich, St. Louis, MO, 200 mM stock in water), 5 mM NaOH, and
buffer (as described, default is 50 mM bicine (Sigma-Aldrich, St.
Louis, MO), 75 mM NaCl, pH 8.5 adjusted with NaOH) and rotating the
mixture overnight at room temperature at 6 rpm. Before the experiments,
the vesicles were filtered over a short (1 cm) Sepharose 4B (Sigma-Aldrich,
St. Louis, MO) size exclusion column and then diluted 1:3 in buffer
containing 1 mM unlabeled oleic acid vesicles (containing neither
biotinylated lipid nor HPTS) to achieve optimal binding density.
Micelles were prepared by mixing 1 equiv of NaOH with 1 equiv of
liquid oleic acid in water and rotating the solution overnight at
room temperature (default concentration: 8 mM).
vesicles were prepared
by mixing 10 mM liquid oleic acid (Nu-Check, Elysian, MN), 20 nM biotin-PEG(5K)-DSPE
(biotin–poly(ethylene glycol)–distearoylglycerophosphoethanolamine,
Nanocs, Boston, MA, 100 μM stock in water), 10 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonate,
Sigma-Aldrich, St. Louis, MO, 200 mM stock in water), 5 mM NaOH, and
buffer (as described, default is 50 mM bicine (Sigma-Aldrich, St.
Louis, MO), 75 mM NaCl, pH 8.5 adjusted with NaOH) and rotating the
mixture overnight at room temperature at 6 rpm. Before the experiments,
the vesicles were filtered over a short (1 cm) Sepharose 4B (Sigma-Aldrich,
St. Louis, MO) size exclusion column and then diluted 1:3 in buffer
containing 1 mM unlabeled oleic acid vesicles (containing neither
biotinylated lipid nor HPTS) to achieve optimal binding density.
Micelles were prepared by mixing 1 equiv of NaOH with 1 equiv of
liquid oleic acid in water and rotating the solution overnight at
room temperature (default concentration: 8 mM).
10
Manganese-Enhanced Imaging of CVN-AD Mice
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