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Automatic instrument compensation was manually fine-tuned on split linear-log dot plots to match median fluorescence values of negative and positive subpopulations.
For each integrin and cell line, the median fluorescence values were taken from gated regions of 10 000 live cells using a FACScan (Becton Dickinson, Franklin Lakes, NJ).
For analysis, standard curves were plotted for median fluorescence values of both the OG-negative and OG-positive buffers.
The data were fitted to calibration curves constructed with the median fluorescence values for each replicate of the standards.
As in Figures 6A and 6B, the cluster sizes were more variable than the median fluorescence values of each cluster (note different scale for heat maps).
Because the distribution of the median intensity is normal, we determined ± 2 SD of the distribution to define the normal median fluorescence values from a normal range between channels 159 and 178.
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A plot combining the signal to background information for all the species was created by normalizing the values for each individual species to the median fluorescence value and then averaging the normalized values.
HN5 (median fluorescence value 3265) and Cal27 (median fluorescence value 1049) expressed the highest amounts of EGFR by FACS and western blot.
Shifts in median fluorescence value (MFV) of treated samples versus the corresponding controls were analyzed, and MFVs of untreated controls were defined as 100%.
Only those traces that contain at least one fluorescence value greater than two times the median fluorescence value during the perfusion with Ca2+-free KRH were considered for analysis of intracellular Ca2+ release.
The logic threshold (i.e., the fluorescence level at which the gate output switches between OFF and ON) is taken to be the median fluorescence value within the threshold window range and is indicated by the red dashed line in Figure 2b,c.
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