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semiquantitative analysis showed the percentage of Nav1.8 immunofluorescent positive (>20% maximum intensity) cells in small, medium and large DRG neurons.
To measure total cell fluorescence intensity, cells were fixed with paraformaldehyde (3.7%, RT, 15 min) and permeabilized with 0.1% Triton X-100.
To assess quenching efficacy and total surface intensity, cells were labelled on ice for 30 min and either quenched or left unquenched.
βcat fluorescent intensity: Cells were outlined, mean intensity measured, background subtracted, and βcat average intensity of a transfected cell normalized to mean of the βcat intensity of 2 3 adjacent untransfected cells.
For analysis of fluorescence intensity, cells were collected and resuspended in 300 μl of 0·5 % bovine serum albumin in PBS and detected using a FACSCalibur flow cytometer and CellQuest Pro software (Becton Dickinson).
We observed that, whereas almost all undifferentiated ESCs expressed Oct4-GFP at high intensity, cells with low-intensity GFP expression were formed by day 14 of differentiation along with a reduction in the percentage of cells expressing GFP (Fig. 1B).
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Fluorescence microscopy allows for good contrast between the bright, but of possibly heterogeneous intensity, cell and the dark background.
According to the fluorescence intensities, cells bound by BA-hBD3 and BA-Hst 5 were only 10 15% of that found for BA-LL37-treated cells (Fig. 4C).
The mean intensity of the high-staining subpopulation was about twice the mean of the low-intensity cells.
Based on these intensities, cells were sorted into four fractions (orange, blue, purple, red) by FACS.
Average ± SEM of normalized mean intensity per cell (n=11 cells, Bem1; n=14 cells, Cdc24).
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