Fri Feb 26, 2016 4:38 pm by mleipold
Hi Osh,
Could you show your gating pathway starting from Ungated (raw) cells? For your PBMCs as well?
I'm trying to eliminate any other issues in your Cell ID staining (Ir staining, Event length, etc). I have attached an example, from Ungated through Live Intact Singlets. In parallel, I included CD3+CD20+ gate examples for the same populations.
I asked about CD3+CD20+ plots as a QC example: if you haven't gated out the majority of your debris, doublets, and dead cells, you will have a lot more cell events that aren't biologically possible (eg, something that expresses both CD3 and CD20). It's one quick way to look at the overall efficiency of your top gates: If you see more than a few CD3+CD20+, or CD14+CD20+, or CD3+CD14+ cells, then you immediately know something isn't right about your gating, staining, or sample acquisition. Especially since you said that the cisplatin seems to be working well for PBMCs. Ideally, if you compare the CD3+CD20+ frequency of your PBMCs to your uterine samples as you go through your top gates, you should get around the same percentage of CD3+CD20+ after you gate out all the debris/doublets/dead in both tissues. By adjusting the Live gate to include the Cisplatin-dim population, it should allow you to figure out whether it is enriched in CD3+CD20+ and *should* be discarded, or whether it has no effect on CD3+CD20+ and therefore is probably Live.
In fact, if there's a question about Live-dead staining, it's a cheaper experiment to just use CD3, CD20, CD14, Live-dead, and Ir, and make sure that your "impossible" populations like CD3+CD20+ are under control before going back to your full panel.
Mike