Wed Mar 19, 2014 2:56 pm by AdeebR
Hi Ofir,
Thanks for sharing your thoughts.
You are correct in that these events are triple positive for the three markers.
However, I realized that my initial post might have misrepresented the experiment a bit - the sample in this case were stained with a 20-parameter staining panel, it just so happens that these three channels are the only ones where I see this strange signal.
Based on this, I don't think this can be explained by doublets/cell clumps (though I will certainly try a tighter gate to see if it helps), since doublets would presumably appear in multiple channels and not just these three. Also, the events are not positive for other markers (e.g. CD66b) which would be expected if they were true clumps of CD16+ neutrophils.
I should also note that this sample was run at <300 events/s.
Regarding your third point, I agree that this exclusion gate seems like it's excluding single positive events, and the rationale for this exclusion is simply that they don't look right (which admittedly is not the most robust reason). However, I know which cells are supposed to express CD123 in the sample (basophils and pDCs), and some of these events are several logs brighter than the true CD123+ population, which doesn't make biological sense. I've also come across these Eu bright events in multiple panels using different sample types and antibodies on the Eu151 channel, which suggests that these are not a true population. I certainly think that it would be nice to have a better explanation as to what these events are.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC