ThT was added to each of the peptide solutions (20 mM (GA)3, (GP)3 and (GR)3 and 10mM (GA)6, (GP)6 and (GR)6 dissolved in 25mM sodium phosphate pH 7.4, 0.1M NaCl) at a final concentration of 10μM, and 110 μl of this mixture was dispensed into each well of Falcon 96-well plate (black/clear, flat bottom, Corning, 353219). Plates were then incubated at 37°C in FLUOstar Omega (BMG Labtech Inc) and were shaken at 200rpm using meander corner well shaking mode. Fluorescence was measured with gain set at 90%, an excitation wavelength of 440 nm and emission wavelength of 490 nm. Four separate replicates were measured for each sample.
Falcon 96 well plate
Manufactured by Corning
Sourced in United States
The Falcon 96-well plate is a laboratory equipment product designed for a variety of cell culture and assay applications. The plate has 96 individual wells, each with a flat-bottom configuration, and is made of polystyrene material. The product is intended to provide a standardized, consistent, and reliable platform for conducting experiments and analyses that require multiple sample wells.
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Lab products found in correlation
3 protocols using falcon 96 well plate
1
Amyloid Fibril Formation Kinetics
2
Thioflavin T Assay for Protein Aggregation
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ThT assays were carried out with 10 μM protein in 0.1 M NaCl, 1 mM sodium azide, and 20 mM Na phosphate pH 7.5. Prior to the assay, proteins were filtered through a 0.22 μm filter. ThT was added to a final concentration of 10 μM. Teflon beads were added into each well of a Falcon 96-well plate (black/clear, flat bottom, Corning, cat # 353219). Then 75 μl of sample were pipetted into each well.
Plates were then incubated at 37°C in a FLUOstar Omega (BMG Labtech Inc) by shaking at 200 rpm using the 'meander corner well shaking' mode. Fluorescence was measured with gain set at 90%, an excitation wavelength of 440 nm and emission wavelength of 490 nm. Three technical replicates were measured per sample for a single ThT assay. Data shown in Fig. 1 are averaged over three to four independent experiments done with different protein preparations. Half times (50% steady-state transition) were calculated as described by 39 .
Plates were then incubated at 37°C in a FLUOstar Omega (BMG Labtech Inc) by shaking at 200 rpm using the 'meander corner well shaking' mode. Fluorescence was measured with gain set at 90%, an excitation wavelength of 440 nm and emission wavelength of 490 nm. Three technical replicates were measured per sample for a single ThT assay. Data shown in Fig. 1 are averaged over three to four independent experiments done with different protein preparations. Half times (50% steady-state transition) were calculated as described by 39 .
3
Evaluating 3D Culture Drug Effects
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Recent studies suggest that the three-dimensional (3D) culture system may provide a better tool to mimic physiological drug function [16, (link)17] (link). The growth-inhibitory effects of YM155 and chrysin were evaluated in the collagen-gel-embedded 3D culture, using a collagen gel culture kit (Nitta Gelatin, Osaka, Japan).
Collagen gel was constituted by a mixture of Cellmatrix I-A, 10 times Ham's F-12 medium, and reconstruction buffer at a ratio of 8: 1: 1. Cells were suspended in the collagen gel at a density of 2 x 10 5 cells/ml, and 50 µl of the gel was injected into each well of a Falcon 96-well plate (Corning, Corning, NY, USA). After semi-solidi cation of the gel by incubation at 37°C for 20 minutes, 40 µl of RPMI1640 medium with 20% FBS was added in each well. After one-day incubation, 10 µl of medium containing serially diluted YM155 and/or chrysin at 4 µg/ml was added onto each well. After incubation for 72 hours, cell survival was evaluated by the CellTiter-Glo luminescent assay.
Collagen gel was constituted by a mixture of Cellmatrix I-A, 10 times Ham's F-12 medium, and reconstruction buffer at a ratio of 8: 1: 1. Cells were suspended in the collagen gel at a density of 2 x 10 5 cells/ml, and 50 µl of the gel was injected into each well of a Falcon 96-well plate (Corning, Corning, NY, USA). After semi-solidi cation of the gel by incubation at 37°C for 20 minutes, 40 µl of RPMI1640 medium with 20% FBS was added in each well. After one-day incubation, 10 µl of medium containing serially diluted YM155 and/or chrysin at 4 µg/ml was added onto each well. After incubation for 72 hours, cell survival was evaluated by the CellTiter-Glo luminescent assay.
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