AC10 human ventricular cardiomyocytes (PTA-1501) and DMEM:F12 medium (supplemented with 2.5 mM glutamine, 15 mM HEPES, 0.5mM sodium pyruvate, and 1200 mg/L sodium bicarbonate) were obtained from the ATCC (Manassas, VA). Characterized fetal bovine serum (FBS) and Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ (PBS), and thioflavin T (ThT) were purchased from Thermo Scientific (Waltham, MA). Hank’s balanced salt solution, 100x penicillin-streptomycin solution, L-glutamine, trypsin, and EDTA were purchased from Corning Life Sciences (Tewksbury, MA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dichloro-dihydro-fluorescein diacetate (DCFH-DA), elastin, crystal violet, and anhydrous dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Cellular ATP quantitation was performed with the CellTiter-Glo Luminescent Cell Viability Assay kit from Promega (Madison, WI) according to the manufacturer’s directions. Hoescht 33342, Alexa Fluor 488 NHS Ester (succinimidyl ester), and Alexa Fluor 647 phalloidin were purchased from Life Technologies (Grand Island, NY). BD Cytofix was from Becton-Dickinson (Franklin Lakes, NJ). 5-chloromethylfluorescein diacetate (CMFDA) was purchased from Genecopoeia (Rockville, MD).
Thioflavin t tht
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
Thioflavin T (ThT) is a fluorescent dye used in the detection and quantification of amyloid fibrils. It binds to the cross-β-sheet structure of amyloid aggregates, resulting in an enhanced fluorescence signal. ThT is a commonly used tool in the study of protein aggregation and amyloid-related diseases.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (1 mentions)
Cytofix, by BD (1 mentions)
Hoescht 33342, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by American Type Culture Collection (1 mentions)
Hank s balanced salt solution, by Corning (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Anhydrous dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions)
L glutamine, by Corning (1 mentions)
Trypsin, by Corning (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Corning (1 mentions)
Cell culture slides, by MatTek (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Abcam (1 mentions)
3 aminopropyltriethoxysilane, by Merck Group (1 mentions)
Cell fractionation kit, by Cell Signaling Technology (1 mentions)
Aβ peptide, by AnaSpec (1 mentions)
Tween 20, by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
8 protocols using thioflavin t tht
1
Characterization of Human Ventricular Cardiomyocytes
2
Fluorescent Amyloid-Beta Nanoparticle Synthesis
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Tetraethyl orthosilicate, poly(N-isopropylacrylamide), ammonium hydroxide, dimethyl formamide (DMF), fluorescein isothiocyanate (FITC), Tween 20, 3-aminopropyl triethoxy silane (Sigma-Aldrich, St. Louis, MO), Calcein AM (CAM) (MilliporeSigma, Burlington, MA), polystyrene (PS) spheres (PolySciences, Warrington, PA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), rhodamine-labeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (Rh-PE) (Avanti Polar Lipids, Alabaster, AL), Opti-MEM (OM) reduced serum media, phosphate saline buffer (PBS), Thioflavin T (ThT) (Thermo Fisher Scientific, Waltham, MA), cell culture slides (MatTek, Ashland, MA), Aβ peptide (AnaSpec, Fremont, CA), biotinylated Aβ antibody (Mabtech, Stockholm, Sweden), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Abcam, Cambridge, UK), and Cell Fractionation Kit by Cell Signaling Technology (Danvers, MA) were used.
3
Thioflavin-T Fluorescence Assay
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Thioflavin-T (ThT) was obtained from Alfa Aesar (Ward Hill, MA). Synthetic α-syn (140 amino acids) was obtained from rPeptide (Watkinsville, GA). Synthetic α-syn fragment peptides of 25 amino acids were purchased from GenScript (Piscataway, New Jersey).
4
Signal Peptide Preparation and Characterization
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All signal peptides used in the study were prepared and obtained from GenScript USA Inc (Piscataway, NJ). Hexafluoroisopropanol (HFIP) and thioflavin T (ThT) were purchased from Alfa Aesar (Ward Hill, MA).
5
Fibrillization Assay of α-Synuclein
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A fibrillization assay was performed using 100 μL of 1 mg/mL monomeric αSYN (rPeptide). Samples containing either αSYN alone or αSYN + NPs at 1:10 volume ratio were loaded with 20 μM Thioflavin T (ThT) (Acros Organics) into 96 well clear bottomed plates (Corning), sealed with Axygen sealing tape (Corning), and shaken at 600 rpm at 37°C for at least 63 h. Samples were taken at various time points and a POLARstar Omega plate reader (BMG Labtech) was used to monitor the increase in ThT intensity, with fluorescence intensity measured at excitation −450 nm and emission −485 nm. This protocol was adapted from the literature (Moriarty et al., 2017 (link)).
6
Lysozyme Amyloid Fibrillation Assay
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Hen egg white lysozyme (L6876) was obtained from Sigma-Aldrich (Saint Louis, MO, USA) and used directly. Sodium chloride (NaCl) with >99% purity was purchased from Sigma Aldrich (Saint Louis, MO, USA). Tannic acid in analytical grade was purchased from Aladdin (Shanghai, China). Hydrochloride acid (HCl) in analytical grade with a purity of 36–38% was purchased from Kemiou Chemical Reagent (Tianjin, China). Thioflavin T (ThT) was purchased from Acros (Geel, Belgium). No further purification was performed on all the chemicals. Deionized water with a resistivity of 18.2 MΩ·cm was obtained from a Millipore system (Billerica, MA, USA). Mica (muscovite) was obtained from a local vendor.
7
Fluorescence Assay for Amyloid Aggregation
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Thioflavin T (ThT) was purchased from Acros Organics (Fair Lawn, NJ). Lipids [dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC)] were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). The 96-well plates (black, w/flat bottom) were bought from Greiner Bio-One (Monroe, NC). All the chemicals were purchased from Sigma Aldrich (St. Louis, MI) and used without further purification. Silica plates (w/UV254, aluminum backed, 200 micron) and silica gel (standard grade, particle size = 40–63 micron, 230 × 400 mesh) for flash column chromatography were purchased from Sorbent Technologies (Atlanta, GA). Dry solvents were purchased from Sigma Aldrich (St. Louis, MI). Human islet amyloid polypeptide was purchased from Anaspec (Fremont, CA) with >98% purity. IAPP was re-purified using in-house purification method.
8
Characterization of Amyloid-β Peptides
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Fine chemicals were procured as follows: Thioflavin T (Th T) from Acros Organics, Belgium; 8anilino-1-naphthalene sulfonic acid (ANS), 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and Paraformaldehyde (PFA) from Sigma-Aldrich, St. Louis, Missouri, USA; Dimethyl sulfoxide (DMSO) from HiMedia Laboratories, Mumbai, India; trypsin (3x crystallized, bovine pancreas), carboxypeptidase (bovine pancreas, type II -PMSF treated aqueous suspension), α-chymotrypsin (hog pancreas), elastase (porcine pancreas) and pepsin from SRL, Mumbai, India; acetic acid, formic acid, Acetonitrile (ACN) and Trifluoroacetic acid (TFA) from Merck, Germany; urethane from AMRESCO, Texas, USA; uranyl acetate from BDH Corporation, Mumbai, India; copper grids (300-mesh size) and mica sheets (size 20620 MM, 0.27 -0.33 mm thickness) from Electron Microscopy Sciences, Pennsylvania, USA; MTT from Molecular Probes, Invitrogen, USA; Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, trypsin-EDTA and Fetal Bovine Serum (FBS) from Gibco, Maryland, USA. HPLC-purified peptides Aβ40/42 were procured from American Peptide, Sunnyvale, CA, USA. Physical and chemical homogeneity of monomeric Aβ peptide was verified by mass spectrometric analysis where other than trace amount of dimer, trimer and tetramer of the peptide, no other impurities could be detected.
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