Wed Mar 18, 2020 4:18 pm by mleipold
Hi Komal,
You've shown us rain plots and FlowJo workspace gating pathway screenshots, but you haven't shown us any bivarate plots.
So, let's back up a few steps: if you take your original Raw files (before concatenation, before normalization), put them into FlowJo, and then look at Ir vs Ce140, do you *see* EQ beads in the bivariate plot?
If so, then repeat for your normalized file(s) and repeat also for your concatenated file (or whichever order you did those in this time).
In other words:
1. Find out if the beads are actually "missing", or if your gate is just "off". Both would result in zero beads in your gate.
2. If the beads are actually missing, then you would find out which step made them disappear. For example, most normalization methods allow you to "remove" the EQ beads in the final step, so if that box accidentally got checked, your files wouldn't contain them. Or, if the normalization somehow got screwed up, your beads would still be there, but would be outside of your gate.
Mike