To prepare seeds, FVIII samples (0.122 μM) were incubated for 2, 5, 8 or 18 h at 45°C in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan). Native FVIII samples (0.122 μM) were mixed 1:1 with the corresponding seeds and time-dependent aggregation at 45°C was initiated. Samples were stored at −80°C until HPLC-SEC analysis.
Polystyrene microplates
Manufactured by Corning
Sourced in United States
Polystyrene microplates are a type of laboratory equipment used for various applications, including cell culture, enzyme-linked immunosorbent assays (ELISAs), and other assays that require the use of small, individual sample wells. These plates are made of polystyrene, a common plastic material, and are designed to provide a standardized and consistent platform for conducting experiments or tests.
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Lab products found in correlation
Plate thermoshaker, by Biosan (2 mentions)
Synergy h4 hybrid reader, by Agilent Technologies (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Bio-Rad (1 mentions)
Prism 7, by GraphPad (1 mentions)
Ez yeast transformation kit, by Zymo Research (1 mentions)
Bovine serum albumin (bsa), by LGC (1 mentions)
Flexstation 2, by Molecular Devices (1 mentions)
Multimode plate reader, by Agilent Technologies (1 mentions)
Biotek synergy neo, by Agilent Technologies (1 mentions)
One glo ex luciferase assay system, by Promega (1 mentions)
Prism version 8, by GraphPad (1 mentions)
Hek293 cells, by Corning (1 mentions)
Sars cov 2 rbd protein, by Sino Biological (1 mentions)
Horseradish peroxidase hrp conjugated anti human igg antibody, by Abcam (1 mentions)
Prism 8, by GraphPad (1 mentions)
Tmb single component substrate solution, by Solarbio (1 mentions)
10 protocols using polystyrene microplates
1
FVIII Aggregation Kinetics Evaluation
2
FVIII Thermal Stability Assay
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All FVIII samples with protein concentrations of either 0.122 μM for high-performance size-exclusion chromatography (HPLC-SEC) or 0.61 μM for dynamic light scattering (DLS) were incubated at 25, 30, 35, 40, 45 or 50°C for 20 h in polystyrene microplates (Corning Incorporated – Life Sciences, Tewksbury, MA, USA) covered with plate sealers in a Synergy H4 Hybrid Reader (BioTek, Winooski, VT, USA) with 20 s medium shaking every 10 min. Samples were subsequently frozen at −80°C until analysis.
3
Polystyrene Microplate ELISA for HA Binding
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Polystyrene microplates (Corning Inc., Corning, NY, USA) were coated overnight at 4 °C with 1 µg/mL rFL HA in carbonate-bicarbonate buffer pH 9.6, then washed three times with PBS containing 0.05% Tween-20 (TPBS), and incubated for 1 h with blocking buffer (TPBS containing 1% w/v casein) at 37 °C. After that, plates were rinsed three times with TPBS and VHH-Fc (serial dilutions in blocking buffer) were added to the wells and incubated for 1 h at 37 °C. Immunoplates were washed four times and bound antibodies were detected using polyclonal goat anti-human IgG HRP-conjugated antibodies (MilliporeSigma, Burlington, MA, USA), diluted 1:20,000 in blocking buffer. After five washes, 3,3′5,5′-tetramethylbenzidine (TMB) (Bio-Rad, Hercules, CA, USA) was added as a substrate. Fifteen minutes later, the reaction was stopped by the addition of 1 M H2SO4 and the absorbance was read at 450 nm. The half maximal effective concentration (EC50) values were calculated using four-parameter logistic regression using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA).
The antibody binding activity to untreated and denatured HA was measured as described above. rFL HA subtype H1 was denatured in 0.1% SDS, 50 mM DTT by heating at a dry block thermostat at 99 °C for 10 min.
The antibody binding activity to untreated and denatured HA was measured as described above. rFL HA subtype H1 was denatured in 0.1% SDS, 50 mM DTT by heating at a dry block thermostat at 99 °C for 10 min.
4
Yeast Transformation Efficiency Evaluation
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Competent cells of each yeast strain were prepared by EZ-yeast Transformation kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions, and their transformation efficiency was assessed for each introduced plasmid. Equal amounts of yeast transformants that were adjusted according to the transformation efficiency were directly suspended in 4 mL SDC+ 5 × U liquid medium and cultured at 250 rpm and 30 °C for 48 h. The harvested cells were washed with 4 mL of PBS (10 mM Na2HPO4, 1.76 mM NaH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) twice and then diluted to 0.2 of OD600 in 200 μL SDC liquid medium on the 96-well Polystyrene Microplates (353072; Corning, Corning, NY, USA). During the incubation at 30 °C without shaking, the cell growth curve was monitored by measuring absorbance at 600 nm of cell culture with the plate reader (Tecan Infinite 200 Pro F Plex; Tecan Ltd., Männedorf, Switzerland). The plates were sealed tightly by the transparent sealing tape (232698; Thermo Fisher Scientific), and the moat was filled by 70 μL of sterilized 0.1% agarose (Nacalai tesque) to reduce evaporation of the medium. The maximum specific growth rate was calculated by using Curveball 0.2.16 [39 (link)].
5
FVIII Thermal Stability Assay
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All FVIII samples (0.122 μM) were incubated at 45°C for 24 h in polystyrene microplates (Corning) covered with plate sealers in a plate thermo shaker (Biosan, Riga, Latvia). Samples were withdrawn after various time intervals and immediately frozen at −80°C until HPLC-SEC analysis.
6
ZIKV and YFV Viral Particle ELISA
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Polystyrene microplates (Corning, New York, NY, EUA) were coated overnight at 4°C with 105 ZIKV or YFV viral particles. Following blocking for 2 h with PBS containing 1% bovine serum albumin (BSA) (LGC Biotecnologia, Cotia, SP), the serum from mice that were vaccinated with YFV was adsorbed in the wells at different concentrations and incubated overnight at 4°C. Then, peroxidase-conjugated goat anti-mouse IgG antiserum (1:4,000; Southern Biotech) was added to the wells, and the plate was incubated for an additional period of 1 h. Peroxidase activity was revealed using hydrogen peroxide and tetramethylbenzidine (TMB). The reaction was stopped with H2SO4 (2.5 N), and the optical density (OD) at 450 nm was determined with a spectrophotometer using SOFTmax PRO 4.0 software (Life Sciences Edition; Molecular Devices Corporation, Sunnyvale, CA).
7
Mitochondrial Membrane Potential Measurement
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∆Ψm values were measured using 5,5′,6,6′ tetrachloro-1,19,3,39-tetraethylbenzimidazoyl-carbocyanine iodide (JC1, catalog no. T3168, Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA), a lipophilic cationic dye. Due to positive charges and lipophilic characteristics, JC-1 monomers concentrate to mitochondria where the membrane potentials are extremely negative (about −150 to −200 mV) and aggregate into polymers. While the monomers were excited at a wavelength of 490 nm and emitted 535 nm green fluorescence, the excitation and emission wavelengths of the polymers were 535 nm and 590 nm (red fluorescence), respectively.
Brown adipocytes were seeded in 96-well black polystyrene microplates (catalog no. 3603, Corning, New York, NY, USA) a day before the experiment. The cells were incubated with 2 μM JC-1 in KRB solution for 30 min and then changed to T3 solution (in KRB) for 30 min before the measurement. The fluorescence signals were recorded by a fluorescence microplate reader (FlexStation II, Molecular Devices, San Jose, CA, USA). The ∆Ψm were estimated by a ratio of red/green (590 nm/535 nm).
Brown adipocytes were seeded in 96-well black polystyrene microplates (catalog no. 3603, Corning, New York, NY, USA) a day before the experiment. The cells were incubated with 2 μM JC-1 in KRB solution for 30 min and then changed to T3 solution (in KRB) for 30 min before the measurement. The fluorescence signals were recorded by a fluorescence microplate reader (FlexStation II, Molecular Devices, San Jose, CA, USA). The ∆Ψm were estimated by a ratio of red/green (590 nm/535 nm).
8
Fluorogenic Peptide-Based Protease Assay
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The assay of protease activity of purified NS2B(H)-NS3(pro) was carried out using previously reported fluorogenic peptides from JEV conserved cleavage sites NS2B/NS3 (Pyr-RTKR-amc) and NS2A/NS2B (Dabcyl-PNKKRGWP-(EDANS)G) synthesized by A+Peptide (Pudong, Shanghai, China) [42 (link)]. Assays were conducted on 96-well black, flat bottom, tissue culture treated polystyrene microplates (Corning Life Sciences, MA, USA) in total reaction volume of 0.1 ml containing 0.5 mM NS2B(H)-NS3(pro) proteases, assay buffer (50mM Tris HCl, pH9.5, 20% glycerol) and substrate. For kinetic assessments, fluorescence measurements were recorded at 20 minutes interval for 520 minutes through Multimode plate reader (Bio Tek) at an excitation wavelength(λex) of 360nm and emission wavelength(λem) of 460 nm. Assay was carried out with three repeats for each group in two independent experiments at a constant temperature of 37°C. Inner filter effects of microplate reader were corrected as described in literature [46 (link)]. To determine the amount of AMC/fluorescence released, a standard curve was plotted with various value of kinetic data. No significant hydrolysis of peptide substrate was noted in dead NS2B(H)-NS3(pro) and/or groups without enzymes. Data was analyzed using GraphPad Prism version 7.00 for windows (GraphPad Software, La Jolla, California, USA)[47 , 48 (link)].
9
ACE2-Mediated SARS-CoV-2 Pseudovirus Entry Inhibition
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HEK 293 cells (ATCC® CRL-1573) stably over-expressing full-length human ACE2 protein were seeded in 96-well white polystyrene microplates (Corning, Cat. No. CLS3610) at 0.03 × 106 cells/well in DMEM (10% FBS and 1% Pen-Strep), and grown overnight at 37 °C, 5% CO2. To test the inhibition of pseudovirus entry by ACE2 WT or mutants, increasing concentration of Fc-tagged ACE2 proteins were first mixed with pseudoviruses and incubated at room temperature for 10 m72 . The ACE2 WT or mutant treated pseudovirus mixture was then used to infect cells. The cells were incubated at 37 °C, 5% CO2 for 6 h, then the medium was replaced with fresh DMEM (10% FBS and 1% Pen-Strep), and then again every 24 h for up to 72 h. To measure the luciferase signal (a proxy for virus uptake), DMEM was removed and cells were replaced in DPBS (ThermoFisher, Cat. No. 14190250) and mixed with an equal volume of ONE-Glo™ EX Luciferase Assay System (Promega, Cat. No. 8130). Relative luciferase units were measured using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data was then analyzed using GraphPad Prism Version 8.4.3 (GraphPad Software, LLC.).
10
SARS-CoV-2 RBD Protein ELISA
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ELISA was performed according to published protocol21 (link). Polystyrene microplates (Corning) were coated overnight with 2 μg/mL SARS-CoV-2 RBD protein (Sino Biological). After washed with PBS containing 0.2% Tween 20 (Solarbio Life Sciences), the plates were blocked using 2% BSA (Sigma Aldrich) in PBST for 1 h at 37 °C. Following washing with PBST, serial dilutions of testing antibodies were added to each well and incubated at 37 °C for 1 h. After washing with PBST, horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (Abcam) was added at the dilution of 1:10,000 and incubated at 37 °C for 1 h. After washing, TMB singlecomponent substrate solution (Solarbio Life Sciences) was added to the microplate and incubated at room temperature for 6 min, followed by adding 2 M H2SO4 to stop the reaction. The absorbance was detected at 450 nm/630 nm. The data was analyzed using GraphPad Prism 8.0.
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