CyTOForum

So I recently normalized about 50 FCS files together and noticed that the intensities of the beads after normalization are not all the same for each file (ie each colored line is not a horizontal line). Is this something that is normal?

Could you attach the bead-normalizer before/after picture that you received? Just in case there is an issue.

However: the MATLAB normalizer (Finck et al: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3688049/) is *not* like the Fluidigm normalizer (which *will* give you perfectly overlapping bead signal intensities, assuming you use Passport_ver2).

If you look at Fig 2E, Fig 6A (bottom) or Fig 6C (bottom) of the Finck et al paper, you see that even after normalization, the beads are much closer than before, but not perfectly overlapping.


Remember:
MATLAB essentially takes the local value per file, then makes a global value from all the files together, then readjusts each file onto the global value. This won't give you perfect overlap, but *will* allow for differences in machine mass sensitivity, etc to remain.
Fluidigm uses an external set of "correct" values (as determined by Fluidigm), and adjusts each file back to those values. This will give you perfect overlap, but will warp any machine-specific mass sensitivity variance, as well as pull down intensities if your machine was more sensitive overall than theirs.


Mike