The screening of the 1,680 compounds in the compound library for antagonist activity at the ZAC-HEK293 cell line was performed in the FMP assay essentially as previously described [33 (link)]. The day before the assay, cells were split into poly-D-lysine-coated black 96-well plates with clear bottoms (BD Biosciences, Palo Alto, CA) (6 × 104 cells/well). The following day, the culture medium was aspirated and the wells were washed once with 100 μl assay buffer (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 11 mM HEPES, 10 mM D-glucose, pH 7.4), after which 100 μl assay buffer supplemented with FMP dye (0.5 mg/ml) and the various test compounds were added to the wells. The plate was then incubated at 37 °C for 30 min and assayed in a NOVOStar plate reader (BMG Labtechnologies, Offenburg, Germany) measuring emission at 560 nm caused by excitation at 530 nm before and up to 1 min after the addition of 33 μl of agonist solution. In the screening, the 1,680 compounds in the compound library were tested at assay concentrations in the 10–30 μM range, using Cu2+ as agonist at an assay concentration of 200 μM.
Poly d lysine coated black 96 well plates with clear bottoms
Manufactured by BD
Poly-D-lysine-coated black 96-well plates with clear bottoms are a type of laboratory equipment used for various cell culture applications. The plates feature a black exterior and a clear bottom, allowing for optical observations and measurements. The surface of the plates is coated with poly-D-lysine, which enhances cell adhesion and promotes cell growth.
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2 protocols using poly d lysine coated black 96 well plates with clear bottoms
1
Screening Compounds for ZAC-HEK293 Antagonist Activity
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Screening Compounds for ZAC-HEK293 Antagonist Activity
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The screening of the 1,680 compounds in the compound library for antagonist activity at the ZAC-HEK293 cell line was performed in the FMP assay essentially as previously described [33 (link)]. The day before the assay, cells were split into poly-D-lysine-coated black 96-well plates with clear bottoms (BD Biosciences, Palo Alto, CA) (6 × 104 cells/well). The following day, the culture medium was aspirated and the wells were washed once with 100 μl assay buffer (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 11 mM HEPES, 10 mM D-glucose, pH 7.4), after which 100 μl assay buffer supplemented with FMP dye (0.5 mg/ml) and the various test compounds were added to the wells. The plate was then incubated at 37 °C for 30 min and assayed in a NOVOStar plate reader (BMG Labtechnologies, Offenburg, Germany) measuring emission at 560 nm caused by excitation at 530 nm before and up to 1 min after the addition of 33 μl of agonist solution. In the screening, the 1,680 compounds in the compound library were tested at assay concentrations in the 10–30 μM range, using Cu2+ as agonist at an assay concentration of 200 μM.
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