Fri Mar 06, 2015 4:46 pm by mleipold
Hi Ian,
I note a few things:
1. If these are 10min injections, then there are some processing issues. 370K (or even 250K) events in 10min is running your samples WAY too fast. You're going to have doublet issues, especially on a CyTOFv1. Your event rate should be closer to 150K, max, in 10min.
2. I notice that your Ir+ events are often less than half of your Ungated events. This argues that you have washing/debris and/or antibody titer problems. If your samples aren't sufficiently washed or your antibody isn't titered well, you'll have higher background in various channels, in the form of speckles/tire tracks in affected channels. Remember, when a cell event is "called" by the algorithm, it grabs the metal signal information from each channel in that event's time window. This means it will grab background from free antibody or debris, even though there's no "true" signal there.
In support of this idea, notice how your CD127 background for CD127-CD3- population decreased in Sample 4 (60% Ir+, 265K Ungated) vs Sample 1 (31% Ir+, 377K Ungated).
Additionally, your Ir signal in Sample 1 is lower than in the other 3 samples: much or most of the Cell Length is driven by Ir staining intensity (since ion cloud size is driven by total metal content, and Ir is usually the highest metal signal in your sample, which helps normalize Cell Length across samples regardless of the antibody staining intensity). This again argues that Sample 1 had a lot of debris, which would have thrown off the Ir/cell ratio and lowered your Ir signal (assuming all these samples were stained with the same Ir dilution stock).
Mike