Blood was collected by submandibular bleeding and analyzed for CBC using Heska HT5 Element analyzer at the University of Michigan In-Vivo Animal Core (Ann Arbor, MI) on a fee-for-service basis. Tissue non-heme iron contents were measured as previously reported (74 (link)). Briefly, tissues were homogenized in a buffer containing 1 M HCl and 10% (wt/vol) trichloroacetic acid and heated for 1 h at 95 °C. Following a high-speed spin, iron was quantitated using a ferrozine solution and compared with a standard. Serum iron, ferritin, and hepcidin concentrations were measured using the Serum Iron Assay Kit (Abcam ab239715), Mouse Ferritin ELISA Kit (Abclonal RK02793), and Hepcidin Murine-Compet ELISA Kit (Intrinsic Lifesciences HMC-001), respectively, according to the manufacturer’s instructions. Copper content in tissues and serum was measured using a Copper Assay Kit (Sigma MAK127) according to the manufacturer’s instructions. Serum CP activity was measured using a Ceruloplasmin Colorimetric Activity Kit (Arbor Assay K035-H1) according to the manufacturer’s instructions.
Copper assay kit
Manufactured by Merck Group
Sourced in United States
The Copper Assay Kit is a laboratory equipment product designed to quantitatively determine the concentration of copper in various samples. It provides a reliable and accurate method for measuring copper levels across a range of applications.
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Lab products found in correlation
Serum iron assay kit, by Abcam (1 mentions)
Vivaspin 500, by Sartorius (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
2 6 dichlorophenolindophenol, by Merck Group (1 mentions)
Phosphate buffer saline (pbs), by Thermo Fisher Scientific (1 mentions)
Nickel 2 chloride, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Mtt assay kit, by Thermo Fisher Scientific (1 mentions)
Luria bertani, by Thermo Fisher Scientific (1 mentions)
Iron 3 hexahydrate, by Merck Group (1 mentions)
Cobalt 2 bromide, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Trizma hydrochloride, by Merck Group (1 mentions)
Copper 2 chloride, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tannic acid, by Merck Group (1 mentions)
Zinc 2 chloride, by Merck Group (1 mentions)
Tyrosinase activity assay kit, by Abcam (1 mentions)
Glucose assay kit, by Abcam (1 mentions)
Lipid peroxidation mda assay kit, by Abcam (1 mentions)
7 protocols using copper assay kit
1
Comprehensive Blood and Tissue Iron Analysis
2
Mannose-Alkyne Diblock Copolymer Synthesis
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Mannose-alkyne
was fabricated as
previously described.23 (link) All diblock copolymers
were fabricated using 4-cyano-4-(ethylsulfanylthiocarbonyl)-sulfanylpentanoic
acid (ECT) as a chain-transfer agent (CTA) conjugated to either azide-PEG
(AzPEG) or PEG. ECT was synthesized as previously described.62 (link),63 (link) The AzPEG and PEG macro-CTAs were then RAFT-polymerized with DMAEMA
and BMA at a 50:50 molar ratio as previously described.23 (link)−25 (link) AzPEGDB was then conjugated with mannose-alkyne via CuAAC chemistry
to produce MnPEGDB. All polymers were characterized using 1H nuclear magnetic resonance (1H NMR) spectroscopy (Bruker,
400 MHz), Fourier transform infrared spectroscopy (FTIR, Bruker Tensor
27), and a copper assay kit (Sigma-Aldrich). All NMR spectra are shown
in Supporting InformationFigures S2–S4 . Details for the polymer synthesis appear in Supporting Information Materials and Methods .
was fabricated as
previously described.23 (link) All diblock copolymers
were fabricated using 4-cyano-4-(ethylsulfanylthiocarbonyl)-sulfanylpentanoic
acid (ECT) as a chain-transfer agent (CTA) conjugated to either azide-PEG
(AzPEG) or PEG. ECT was synthesized as previously described.62 (link),63 (link) The AzPEG and PEG macro-CTAs were then RAFT-polymerized with DMAEMA
and BMA at a 50:50 molar ratio as previously described.23 (link)−25 (link) AzPEGDB was then conjugated with mannose-alkyne via CuAAC chemistry
to produce MnPEGDB. All polymers were characterized using 1H nuclear magnetic resonance (1H NMR) spectroscopy (Bruker,
400 MHz), Fourier transform infrared spectroscopy (FTIR, Bruker Tensor
27), and a copper assay kit (Sigma-Aldrich). All NMR spectra are shown
in Supporting Information
3
LLPS of Prion Protein with Copper
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For sample preparation, the proteins were thawed on ice and centrifuged (20,000g for 10 min at 4 °C). To exchange the buffer to 10 mM Tris, pH 7.4, the solution was centrifuged (five times at 12,000g for 7 min at 4 °C) through Vivaspin 500 columns with 30-kDa molecular weight cut off (Sartorius Stedim biotech). Afterward, protein concentration was determined by NanoDrop 2000. Phase transition was initiated by adding TEV protease for 1 h to the samples. To analyze the effect of copper on PrP phase separation, we used a buffer with high-purity Tris-HCl from PanReac AppliChem. In addition, before every experiment, the protein solutions were tested for copper contamination with the Copper Assay Kit from Sigma-Aldrich. When no copper was detected with this kit, the corresponding protein solution was defined as copper free. To study the effect of increasing copper concentrations on LLPS, CuCl2 was added to the reaction before TEV protease.
4
Fibril Conversion Dynamics of Prion Proteins
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Fibril conversion starting from 0.25 μg/μL PrPWT or PrPΔOct was performed in buffer solution containing 50 mM of 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.5), 2 M of GdnHCl, and 2.5 ng/μL of PrPWT or PrPΔOct fibril seeds at 37 °C under shaking as described previously [33 (link)]. The progress of fibril formation was monitored by fluorescence intensity of thioflavin T (ThT) for its binding to the cross-β-sheets in the fibrils. The fibrils converted from PrPWT and PrPΔOct are denoted as fPrPWT or fPrPΔOct, respectively. The morphology of fPrPWT was examined by transmission electron microscopy (TEM) according to a previously described procedure [33 (link)]. The fibril samples were dialyzed against water and then mixed with CuCl2 or MnCl2 for further experiments. The binding of copper ions to fPrPWT was examined using a Copper Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).
5
Antimicrobial Evaluation of Metal Complexes
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Tannic acid (TA, ACS reagent), titanium (IV) bis (ammonium lactato) dihydroxide (Ti-BALDH) solutions 50 wt% in H2O, iron (III) hexahydrate (FeCl3.6H2O), copper (II) chloride (CuCl2), cobalt (II) bromide (CoBr2), nickel (II) chloride (NiCl2), zinc (II) chloride (ZnCl2), 2,6-dichlorophenolindophenol, trizma hydrochloride (Tris), copper assay kit and peroxidase from horseradish (HRP) lyophilized powder were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS) was obtained from Acros Organics. Luria-Bertani (LB) and tryptic soy broth (TSB) were purchased from Gibco. MTT assay kit and sodium hydroxide (NaOH) were acquired from Thermo Fisher Scientific. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. NIH 3T3 fibroblasts were obtained from Bioresource Collections and Research Center (BCRC), Taiwan. S. epidermidis ATCC12228, methicillin-resistant S. aureus (USA300, ATCC BAA-1718) and E. coli ATCC12435 were acquired from Bioresource Collections and Research Center in Taiwan. All the water used was purified to 18.2 mΩ using a Millipore water purifications system, and filtered using a 0.22 µm filter.
6
Colorimetric Copper Release Assay
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The release of copper from treatment with Cu/chelators was evaluated in PBS using a copper assay kit (Sigma, MAK127). PBS with Cu/chelators was incubated for 10 min with a colorimetric reagent and analyzed at 359 nm with the spectrophotometer. A standard curve was created by plotting the absorbance values of the 100, 200, and 300 µg/dL standards against the concentration of copper in these standards (R2 = 0.99), and a regression equation was used to calculate the amount of copper (μM) in the samples. This assay can detect copper concentrations up to 47 μM.
7
Multiparametric Evaluation of Oxidative Stress
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Tyrosinase concentrations were assayed using (Tyrosinase Activity Assay Kit, Abcam, USA, Cat. No. ab252899) following colorimetric method Qu et al. [16] , Serum copper was determined using (Copper Assay Kit, Sigma-Aldrich , USA, Cat. No. MAK127) following the method of Changfeng et al. [17] . Serum fasting glucose levels were determined using (Glucose Assay Kit, Abcam, USA, Cat. No. ab65333 ) as described by Saw et al. [18] , Total Antioxidant (TAC) in liver tissue was determined using (Total Antioxidant Assay Kit, Elabscience Biochemistry Inc, USA, Cat.
No. E-BC-K136-S) following colorimetric assay of Marziyeh et al. [19] . Malondialdehyde (MDA) in liver tissue was assayed using (Lipid Peroxidation (MDA) Assay Kit, Abcam, USA, Cat. No. ab118970) using a method adapted to Wang et al. [20] . Hemoglobin, White Blood Cells, and Platelet counts were determined by the automatic cell counter as previously described [21] .
No. E-BC-K136-S) following colorimetric assay of Marziyeh et al. [19] . Malondialdehyde (MDA) in liver tissue was assayed using (Lipid Peroxidation (MDA) Assay Kit, Abcam, USA, Cat. No. ab118970) using a method adapted to Wang et al. [20] . Hemoglobin, White Blood Cells, and Platelet counts were determined by the automatic cell counter as previously described [21] .
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