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For pharmacokinetic analysis, plasma sampling was performed during the first course and assayed using a validated high-performance liquid chromatographic assay with mass spectrometric detection.
The accuracy of the method was proven by using spiked solutions that were prepared by spiking in the appropriate amount of analyte of interest into the sample matrix and assayed using a standard.
The sections were then fixed with 4% paraformaldehyde and assayed using a fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit (Roche Applied Science, Mannheim, Germany).
Embryos with GFP expression within the olfactory region were selected for further analyses, stained with a Tuj1 antibody to visualise the nerve tracts [22] and assayed using a whole embryo 3D-OPT imaging technique.
After 48 hours, cell supernatant was removed and assayed using a commercial ELISA kit from R&D Systems (Minneapolis, MN).
Proteins from the orange and pale green clusters were purified and assayed using a library of proline derivatives.
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A library of 3060 mutants of Y. pestis CO99-3015 was made and assayed using an in vitro invasion assay with gentamicin protection.
For plasma ACE2 activity, blood collected into heparinized tubes was centrifuged at 4 °C and assayed using an ACE2-specific QFS.
Where indicated, culture supernatants were collected and assayed using an IL-12 p70 ELISA kit (Mabtech AB, Nacka Strand, Sweden).
For measurements of IFN- γ secretion, after 5 days in culture, supernatants were collected, pooled and assayed using an ELISA kit (Mabtech).
For plasma ACE2 activity, blood collected into heparinized tubes was centrifuged at 4°C and assayed using an ACE2-specific QFS [ 7-methoxycoumarin-4-yl -acetyl-Ala-Pro-Lys (2, 4-dintirophenyl); Auspep] [ 7-methoxycoumarin-4-yl -acetyl-Ala-Pro-Lys
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