Mon Aug 17, 2015 8:05 pm by AdeebR
Mike, yes the parent gate for the CD4 vs IdU plot is the 165lo 169hi gate from the left plot. The parent gate of that plot is just total "cells - beads". I should note that the two CD45 population (169 and 165) were barcoded, which is why the double positive events can be unambiguously assigned as true doublets.
I think some of the cell based heterogeneity is more apparent in tissues than in blood/PBMCs partly because of the cell types involved. I find that PBMCs are usually pretty uniform in DNA content, however with whole blood, granulocytes (neutrophils and eosinophils) certainly have higher Ir193 signal than lymphocytes. However this difference is not as dramatic as the example of tissue macrophages and epithelial cells that I presented in those example plots, and is usually distinguishable from true doublets.
Also, as you noted, these are inherent differences in signal intensity with, I believe, valid biological basis behind them so the front end acquisition system shouldn't make a difference in their incidence. However, the Helios software is also going to be collecting additional features of the ion cloud distribution besides "event length", and in theory additional information about this distribution should potentially allow a better separation of ion cloud fusion events (probably wouldn't make a difference for true cell aggregate doublets). As far as I know, this is still theoretical though.
Bill, the Epcam staining in this example was with mouse, and I was using clone G8.8. The CD68 data was with human tissue and in that case I was using clone Y1/82A.
Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC