When measuring APC labelled protein on neutrophils using the R670 channel on a Fortessa flow cytometer and FACS DIVA software, both the isotype control and unstimulated labelled cells are set to have a median fluorescence of approx 300.
Stimulated cells have a substantially elevated signal, and the range of intensities covers the whole log scale. In order to fit in the high intensity events the negative values are shifted to below zero, rather than being in line with the negative values of the isotype or unstimulated group. This prevents direct comparison of mean/median fluorescence intensities, or quantifying the % of stimulated cells which have a higher value than the unstimulated sample.
Can we reset the software so that the low values are aligned, and the high values will be pushed up against the right hand axis if they exceed the available log scaling? Or increase the available log scaling so that the whole range of measurements will fit?
I don't want to reduce the voltage too much as I need to keep the isotype value above zero.