Native and decellularised tissue samples from each of six different human aortas were fixed in zinc fixative solution (0.5% w/v zinc chloride (VWR Chemicals), 0.5% w/v zinc acetate (VWR Chemicals), in 0.1 M Tris buffer containing 0.05% w/v calcium acetate (Sigma) at pH 7.4) for 48 h, dehydrated, and embedded in paraffin wax using a Leica Tissue Processor (TP1020). Serial sections (6–8 µm) were stained with haematoxylin and eosin (H&E), picrosirius red/Millers elastin (to visualize collagen and elastin) and alcian blue (glycosaminoglycans, GAGs) using standard histological methods. Cell nuclei were visualised using DAPI dye (Sigma Aldrich); briefly, sections of tissue were rehydrated in a graded alcohol series before they were incubated in the dark in 0.1 µg mL−1 DAPI dye in dye buffer (10 mM Tris, 1 mM EDTA, 1 mM sodium chloride in water) for 10 min. The sections were viewed using an upright Zeiss Imager M2 microscope under normal Kohler illumination or under fluorescent illumination using a DAPI filter (λex = 340–380 nm/λem = 435–485 nm) and images were captured digitally using Zen Blue Pro (Zeiss).
Calcium acetate
Manufactured by Merck Group
Sourced in United States, Italy, Germany
Calcium acetate is a chemical compound with the formula Ca(CH3COO)2. It is a white, crystalline solid that is soluble in water and widely used in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Hydrochloric acid (hcl), by Merck Group (4 mentions)
Ammonium acetate, by Merck Group (4 mentions)
Sodium nitrite, by Merck Group (3 mentions)
Sodium nitrate, by Merck Group (3 mentions)
Sodium azide, by Merck Group (3 mentions)
Ammonium chloride, by Merck Group (3 mentions)
Sodium tetraborate, by Merck Group (3 mentions)
Deuterium oxide, by Eurisotop (3 mentions)
Sodium dihydrogen phosphate monohydrate, by Merck Group (3 mentions)
Sodium hydrogen phosphate dihydrate, by Merck Group (3 mentions)
Dibutylamine, by Merck Group (3 mentions)
Sodium alginate, by Merck Group (2 mentions)
Zinc acetate, by Merck Group (2 mentions)
Manganese acetate, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Glacial acetic acid, by Merck Group (2 mentions)
Sulfuric acid, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
18 protocols using calcium acetate
1
Histological Characterization of Human Aortic Tissue
2
Isolation of human fibroblasts from skin biopsies
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PS patient (n = 2) and control (n = 2) human fibroblasts were obtained from skin-punch biopsies at the Clinic for Special Children (CSC) in Lancaster, PA, USA, following informed consent. Skin biopsies were performed following approved Institutional Review Board protocols at the University of Pennsylvania and Temple University (where the study was initiated), and Lancaster General Hospital (Lancaster, PA, USA). Parents provided informed consent before their child’s participation.
Fibroblasts were extracted from tissue samples by incubation in 0.25% Trypsin/EDTA (Gibco) overnight at 4°C. The next day, the epidermis was removed, and dermis was digested with Collagenase P (Roche) buffered in 130 mM sodium chloride (Sigma–Aldrich), 10 mM calcium acetate (Sigma–Aldrich), and 20 mM HEPES buffer for 30 min at 37°C. Then 0.5% Trypsin/EDTA (Gibco) was added, and the mixture was incubated at 37°C for an additional 10 min before neutralization with fibroblast culturing media, composed of DMEM supplemented with 10% FBS (Sigma–Aldrich), 10 mM HEPES buffer, 1% penicillin/streptomycin (10,000 U/ml penicillin, 10 mg/ml streptomycin stock), and 1% fungizone. Fibroblasts were pelleted through centrifugation for 5 min at 1,500 rpm, and the pellet was resuspended in fibroblast culturing media to obtain the desired cells.
Fibroblasts were extracted from tissue samples by incubation in 0.25% Trypsin/EDTA (Gibco) overnight at 4°C. The next day, the epidermis was removed, and dermis was digested with Collagenase P (Roche) buffered in 130 mM sodium chloride (Sigma–Aldrich), 10 mM calcium acetate (Sigma–Aldrich), and 20 mM HEPES buffer for 30 min at 37°C. Then 0.5% Trypsin/EDTA (Gibco) was added, and the mixture was incubated at 37°C for an additional 10 min before neutralization with fibroblast culturing media, composed of DMEM supplemented with 10% FBS (Sigma–Aldrich), 10 mM HEPES buffer, 1% penicillin/streptomycin (10,000 U/ml penicillin, 10 mg/ml streptomycin stock), and 1% fungizone. Fibroblasts were pelleted through centrifugation for 5 min at 1,500 rpm, and the pellet was resuspended in fibroblast culturing media to obtain the desired cells.
3
5-ASA Microcrystalline Cellulose Formulation
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5-ASA was prepared from Iran Najo co. (Iran). Microcrystalline cellulose (Avicel® PH101) was provided by Samisaz (Mashhad, Iran). Calcium acetate and potassium phosphate monobasic were obtained from Sigma (China). Sodium alginate (Mw 80–120 kDa with mannuronate/guluronate ratio of 1.56) was purchased from Sigma-Aldrich (USA). Eudragit® RS PO (Mw ∼150 kDa) was provided from Evonic® Degussa (India Pvt. Ltd., Mumbai, India). All other chemicals, reagents, and solvents were of the highest commercially available analytical grade.
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5-ASA was prepared from Iran Najo co. (Iran). Microcrystalline cellulose (Avicel® PH101) was provided by Samisaz (Mashhad, Iran). Calcium acetate and potassium phosphate monobasic were obtained from Sigma (China). Sodium alginate (Mw 80–120 kDa with mannuronate/guluronate ratio of 1.56) was purchased from Sigma-Aldrich (USA). Eudragit® RS PO (Mw ∼150 kDa) was provided from Evonic® Degussa (India Pvt. Ltd., Mumbai, India). All other chemicals, reagents, and solvents were of the highest commercially available analytical grade.
4
Native IM-MS Analysis of αS Variants
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Native IM-MS experiments were performed
on a Synapt G1 HD mass spectrometer (Waters Corporation, Wilmslow,
UK) with traveling (T-wave) ion mobility and a nano-ESI source using
in-house generated gold- and palladium-coated capillaries. αS
variants were analyzed at a concentration of 20 μM, and spectra
were collected with and without the addition of 500 μM zinc
acetate, manganese acetate, or calcium acetate (Sigma Life Sciences,
Germany) at a ratio of 1:25 αS:metal ion. The unbound peak profiles
for all three variants were taken from an external control without
metal, since the unbound peak was sometimes not visible in spectra
with a 25-fold excess of metal ion. MassLynx V4.1 (Waters Corporation,
Wilmslow, UK) was used for data processing. Instrument parameters
were set at capillary voltage 1.4 kV, source temperature 30 °C,
sampling cone 18 V, extraction cone 1.0 V, trap collision energy 5.0
V, transfer collision energy 2.0 V, trap DC bias 30 V, IM wave velocity
300 m/s, and IM wave height 7.0 V. Gas pressures in the instrument
were trap cell 0.0256 mbar and IM cell 0.36 mbar. The IM data was
calibrated according to the Bush database49 (link) using denatured cytochrome c (charge states 13+ to 19+), myoglobin
(charge states 15+ to 24+), and ubiquitin (charge states 7+ to 13+)
at 10 μM in 50% (v/v) acetonitrile, 0.1% (v/v) formic acid.
on a Synapt G1 HD mass spectrometer (Waters Corporation, Wilmslow,
UK) with traveling (T-wave) ion mobility and a nano-ESI source using
in-house generated gold- and palladium-coated capillaries. αS
variants were analyzed at a concentration of 20 μM, and spectra
were collected with and without the addition of 500 μM zinc
acetate, manganese acetate, or calcium acetate (Sigma Life Sciences,
Germany) at a ratio of 1:25 αS:metal ion. The unbound peak profiles
for all three variants were taken from an external control without
metal, since the unbound peak was sometimes not visible in spectra
with a 25-fold excess of metal ion. MassLynx V4.1 (Waters Corporation,
Wilmslow, UK) was used for data processing. Instrument parameters
were set at capillary voltage 1.4 kV, source temperature 30 °C,
sampling cone 18 V, extraction cone 1.0 V, trap collision energy 5.0
V, transfer collision energy 2.0 V, trap DC bias 30 V, IM wave velocity
300 m/s, and IM wave height 7.0 V. Gas pressures in the instrument
were trap cell 0.0256 mbar and IM cell 0.36 mbar. The IM data was
calibrated according to the Bush database49 (link) using denatured cytochrome c (charge states 13+ to 19+), myoglobin
(charge states 15+ to 24+), and ubiquitin (charge states 7+ to 13+)
at 10 μM in 50% (v/v) acetonitrile, 0.1% (v/v) formic acid.
5
Investigating Calcium-Signaling Mechanisms
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Calcium acetate, CaSR antagonist NPS2143, calcium indicator Fluo 3-AM, and ERK kinase inhibitor U0126 were purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK-8) was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). DMEM/F12 and fetal bovine serum (FBS) were purchased from Gibco BRL. Polyclonal antibodies against β-actin, cyclin D1, ERK1/2, phospho-ERK1/2, and p21 were purchased from Cell Signaling Technology Inc. Polyclonal antibodies against CaSR were purchased from Abcam plc.
6
Wollastonite-Calcium Phosphate Paste
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EXAMPLE 2
A mixture consisting of 50% by weight of wollastonite CaSiO3 (Nordkalk) and 50% by weight of calcium dihydrogenphosphate monohydrate Ca(H2PO4)2.H2O (Budenheim) is prepared. The water is added to the pulverulent mixture thus obtained in an amount representing 50% by weight, with respect to the total amount of pulverulent constituents. The practical duration of use measured for this paste is 0.2 min. The same composition is prepared by adding 3% by weight of calcium acetate (from Sigma Aldrich) as retarder. The amount of water added is also 50% by weight. The practical duration of use measured for the paste comprising the retarder is then 3 min.
7
Fatty Acid and Metal Complex Analysis
8
Kinetics of α-Synuclein Amyloid Formation
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Kinetics of αS
amyloid formation were monitored in a 96-well, nonbinding, flat-bottom,
half-area microplate (Corning, USA; 10438082) containing one Teflon
polyball (1/8″ diameter; Polysciences Europe, Eppelheim, Germany)
per each well of sample. Samples of 100 μL containing 100 μM
αS with 20 μM Thioflavin T in 20 mM ammonium acetate,
pH 7.5, were incubated at 37 °C shaking at 600 rpm in a FLUOstar
omega plate reader (BMG Labtech, Ortenburg, Germany). Fluorescence
intensity was measured by exciting at 440 ± 10 nm and collecting
emission at 482 ± 12 nm using a bandpass filter. For experiments
involving the addition of metal ions, zinc acetate, manganese acetate,
or calcium acetate (Sigma Life Sciences, Germany) was added at a concentration
of 2.5 mM (i.e., 25 fold excess over protein) per well. Results were
blank-corrected using wells containing 20 μM ThT in 20 mM ammonium
acetate, pH 7.5, and normalized to the maximum fluorescence value
of each curve.
amyloid formation were monitored in a 96-well, nonbinding, flat-bottom,
half-area microplate (Corning, USA; 10438082) containing one Teflon
polyball (1/8″ diameter; Polysciences Europe, Eppelheim, Germany)
per each well of sample. Samples of 100 μL containing 100 μM
αS with 20 μM Thioflavin T in 20 mM ammonium acetate,
pH 7.5, were incubated at 37 °C shaking at 600 rpm in a FLUOstar
omega plate reader (BMG Labtech, Ortenburg, Germany). Fluorescence
intensity was measured by exciting at 440 ± 10 nm and collecting
emission at 482 ± 12 nm using a bandpass filter. For experiments
involving the addition of metal ions, zinc acetate, manganese acetate,
or calcium acetate (Sigma Life Sciences, Germany) was added at a concentration
of 2.5 mM (i.e., 25 fold excess over protein) per well. Results were
blank-corrected using wells containing 20 μM ThT in 20 mM ammonium
acetate, pH 7.5, and normalized to the maximum fluorescence value
of each curve.
9
Chitosan-modified Viscose Fabric Characterization
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Viscose fabric “15A23 viscose uni Sandy–white,” as provided by IGR Agence (Celje, Slovenia), was used as starting material. Chitosan with deacetylation degree 75–85% and low molecular weight (50,000–190,000 Da), 37% hydrochloric acid, sodium tripolyphosphate, zinc acetate dehydrate, calcium acetate, sodium chlorite, 0.1 M sodium hydroxide, sodium hydroxide, potassium chloride, and n-heptane were from Sigma-Aldrich (Vienna, Austria), of analytical grade, and used for modification, functionalization, and characterization of viscose fabrics without additional purification. Agar, tryptic soy broth, and yeast extract were from Torlak (Belgrade, Serbia) and used for determination of antibacterial activity of viscose fabrics without additional purification.
10
Magnetic Nanoparticle Hydrogel Synthesis
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Poly(dimethylsiloxane) and SYLGARD184 silicone elastomer kit were from Dow Corning. Soybean oil, sodium alginate, calcium acetate, sodium citrate (citric acid trisodium salt dehydrate), span 80, resazurin sodium salt, phosphate-buffered saline (PBS), penicillin, and streptomycin were purchased from Sigma-Aldrich company (Washington, DC, USA).
FeCl3, FeCl2.4H2O, and NH4OH were purchased from Merck company (Darmstadt, Germany). Minimum essential medium alpha (MEM α) and trypan blue solution 0.4% were bought from Fisher Scientific (Portsmouth, NH, USA). Fetal bovine serum (FBS) and trypsin were purchased from Life Technologies company (Carlsbad, CA, USA). More details on materials specifications are listed inTable 1 .
FeCl3, FeCl2.4H2O, and NH4OH were purchased from Merck company (Darmstadt, Germany). Minimum essential medium alpha (MEM α) and trypan blue solution 0.4% were bought from Fisher Scientific (Portsmouth, NH, USA). Fetal bovine serum (FBS) and trypsin were purchased from Life Technologies company (Carlsbad, CA, USA). More details on materials specifications are listed in
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