Hi Vinicius,
I'm not sure what you mean by "the normalization takes into account the different sensitivity for each EQ beads mass (140,142,151,153,165,171 and 172)".
Regarding customer files: it depends on what the customer asks for. If the customer requests normalization, then we perform it. However, we generally try to be agnostic about the normalization method: most people want the Fluidigm method, because that's "easiest". I personally disagree with the Fluidigm method; I believe that the MATLAB method is better, since it doesn't force your data to conform to an external standard that may not be reflective of your machine.
As an example:
Here, separate aliquots of the same prestained sample (stained, frozen, then thawed similar to Sumatoh et al) were run on different machines: a CyTOFv1, a CyTOFv2, two different Helios instruments, and the same CyTOFv2 after it was upgraded to a Helios. At the top are the EQ bead results. At the bottom are various cell marker signals. At the left is the raw data; center is Fluidigm-ver2 normalized; and right is MATLAB-normalized.
The pink (CyTOFv1) bead profile has a different mass sensitivity profile than the v2 or Helios instruments. Similarly, the different instruments have different signal intensities/sensitivities overall. As you can see, the Fluidigm-ver2 method forces all of them to conform to the same values, in several cases decreasing signal intensity and in the case of the CyTOFv1 data, warping it to fit the mass sensitivity profile. The MATLAB method, on the other hand, just averages the signal intensity, with minimal warping.
Is normalization required in all cases? It depends on what you're asking. If you want to compare signal Medians (fold-change, clustering, etc), then it's probably necessary in all cases. However, if you have a well-designed panel and you're just comparing frequencies, then it's not always needed.
As an example:
Here, the same sample was stained, washed, and then split in two. One aliquot was run first sample of the day, the second aliquot was run last sample of the day (~8hr later). I then divided the Late sample by the Early sample, for both Freq Parent and for Median intensity. Normalization definitely helps make Median intensity values more stable, but Frequency isn't affected nearly as much. I've done this sort of assay at least twice, and it's consistent.
In short: normalization won't really hurt, and it can definitely help. But it's important to choose a normalization method and stick with it, for consistency. And, of course, save a copy of the Raw files in case you decide that you want to switch normalization methods!
Mike