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After 24 hr, we performed the receptor availability assay, and analyzed the fluorescence by flow cytometry.
10.7554/eLife.02950.004 Figure 2. The availability assay labels receptors and ligands that can participate in trans signaling.
In the ligand availability assay, we did not detect a significant increase in Jag1 availability due to Lfng or Mfng expression.
We induced varying levels of ligand expression in the Notch1+Dll1 and Notch1+Jag1 cell lines, and analyzed the cells with the availability assay.
As a negative staining control, we incubated CHO-K1 cells with both the ligand and receptor availability assay reagents to establish background staining levels.
(E and F) The ligand availability assay works similarly, except soluble Notch1ext-Fc fragments bind free ligands on the cell surface.
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The biomarkers were initially selected based on a thorough search of the current diabetes biomarker literature, narrowed based on the availability of assay reagents that could be incorporated with high quality into the assay system, and then further selected using statistical modeling.
Two candidate protein biomarkers were arbitrarily chosen based on fold differences (Table 1), putative roles in the pathogenesis of prostatitis, and the availability of assay reagents for verification using the gold standard in protein quantification, the enzyme-linked immunosorbent assay (ELISA).
Data to inform key questions of vaccine science might allow the design of vaccines to progress to a less empirical stage, for example through availability of assay results associated with vaccine efficacy.
Genes were selected for protein analyses based on significance and possibility of detection due to availability of assay.
Newer biomarkers of interest were selected on the basis of biological relevance to important processes, commercial availability and assay reproducibility.
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