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A standard curve was generated during each assay in the range of concentrations 0 100 μM using NaNO2 (Sigma-Aldrich) as standard.
To quantify EV71 RNA copies, a linear standard curve was also generated during each assay using serial dilutions of an EV71 DNA standard by adjusting the standard to a concentration gradient of 1×10 copies/µL to 1×10 copies/µL.
During each assay (see below, Section 2.2) cells were used at a density of 1000 μL−1.
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In principle, this could be done by a human expert using the knowledge of the design and quality of each particular bioassay (e.g., confidence score assigned during manual curation to each assay in ChEMBL).
More flexibility is allowed in biomarker method validation where during pre-study validation each assay can be evaluated on a case per case basis, with 25% being the default value (30% at the LLOQ) for precision and accuracy.
Cases and controls were arranged in a chequerboard pattern on each plate to ensure even treatment during the assay procedure and each plate included negative controls (with no DNA) and positive controls duplicated on a separate quality control plate.
To calculate the mass of each disc consumed during the assay (amount eaten), the final dry mass of each glass fiber disc was subtracted from its initial dry mass.
Optimal seeding densities were determined for each cell line to assure exponential growth during the assay.
To apply the logistic equation to our data we analyse three subregions within each IncuCyte ZOOM™ image at several time points during the assay.
We collected and analyzed supernatants from each condition during the suppression assay in order to determine sCD25 production at 48 and 96 h time points (Figure 3).
As in the RTPA task, time spent in each side during the CPA assay was assessed using Ethovision.
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