Thu Jun 25, 2015 4:14 pm by mleipold
Hi Chad,
Do you happen to have a screenshot of what the acquisition window looks like as you run the sample?
Like Bill, I'm suspicious that you're not completely fixing the cells, and they're busting in the MilliQ. While MilliQ can cause cells to pellet differently than in PBS, the main source of loss in MilliQ is lysis.
Based on your TIFF, the Ir-bright population in your first gate is only ~25% of total Found events. This makes it likely that either you're not washing out the residual Ir, or you're lysing the cells. If you're lysing, then you should have background in just about every channel you measure. This will show up on the Acquisition screen as vertical streaks of varying density (from speckles to Sharpie-like tire tracks). This will also show up as background in all of your Events. This even includes background in all of your Bead events: take a look at the background levels in all of your Marker channels, in all of your Bead events.
Remember, a Found Cell Event is triggered when *any* of your channels satisfies the Event Length and Sigma parameters. This then grabs across *all* channels for the time-window of that particular cell event....if you have streaking, that time-section of the streak will be captured in the Found Cell data.
Mike