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These results indicate a greater dynamic range for assays using DNA hydrolysis probes in the quantification of low copy templates.
Luciferase activity was expressed as relative light units (RLU) for assays using BG1Luc4E2 cells or as a ratio of luciferase RLU/mg protein for assays using SKBR3 cells.
Control experiments were carried out in parallel at 23°C (for assays using UAS-Shibire ts1 ) or 19°C (for assays using UAS-dTrpA1).
Purified CA enzymes were diluted in assay buffer (50 mM Na2SO4, 50 mM HEPES, 50 mM MgSO4, 0.004% (w v-1) Phenol red, pH 8 for assays using CO2 as a substrate or pH 6 for assays using KHCO3 as a substrate.
For assays using PMNs, bacteria were opsonized in 5% NHS.
For assays using mutant strains, cyclohexanone was diluted 1∶100 in ethanol.
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There are some special requirements for assays used in drug discovery that are related to the nature of the information needed to understand drug action.
For assay using CMC, all enzymes were diluted 5x compared to the Avicel reaction in order to resolve results.
Cells were collected after 48 h for assay using the Dual Luciferase reporter assay system (Promega, Madison, WI, USA).
Phosphate buffers were only used for the assays using ultrapure water.
Freshly isolated CD8+ T cells were used for cytotoxicity assays using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin, USA).
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