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2 protocols using alamarblue hs reagent

1

Synthesis and Characterization of DA^yne and Its Interaction with PDIA3

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All chemicals required for the synthesis of DAyne, preparation of buffers, mushroom tyrosinase (Lot#: SLBZ0022), NBT, Amicon ultra 0.5 mL centrifugal filters (3k) and click reactions were purchased from Sigma-Aldrich unless otherwise stated. NMR solvents were obtained from Cambridge Isotope Laboratories Inc. SH-SY5Y neuroblastoma cells were acquired from ATCC. Cell culture media, additives, and consumables were purchased from Corning. High capacity streptavidin agarose resin, Zeba Spin Desalting Columns (7K MWCO, 0.5 mL), PDIA3 (ERp57) antibody (Cat. no. CL2444), and DAPI were obtained from Thermo Scientific. alamarBlue HS reagent and ProLong Glass were purchased from Invitrogen. BCA kits and C-18 spin columns were bought from Pierce. Mini-PROTEAN TGX Precast Gels, 0.2 μm nitrocellulose, and filter paper transfer stacks were obtained from BioRad. Biotin azide, THPTA, Alexa Fluor 647 azide, and azide agarose beads were purchased from Click Chemistry Tools. REVERT total protein stain kit, Odyssey TBS blocking buffer, and IRDye800CW streptavidin were purchased from LI-COR. Recombinant PDIA3 and PDI inhibitor screening kit (fluorometric) were obtained from BioVision (Cat. no. 7601–100 and K840–100, respectively).
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2

Alamar Blue™ HS Cell Viability Assay

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Cell viability analysis was performed with the Alamar Blue™ HS reagent according to manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA, A50101). Briefly described, a 1/10 dilution of Alamar blue solution was added to each well of 24-wellplates for 2 h at 37 °C. Then, 50 µL of cell supernatant were monitored using a fluorescence microplate reader set up at 560 and 605 nm as excitation and emission wavelengths, respectively.
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