Marcia Woda asks: >1. Is it possible to accurately count the cellular events below the >threshold one is using during acquisition? > The threshold is that level above which the trigger signal has to rise for the instrument to define the beginning of an event; the electronics are therefore essentially blind to what goes on below the threshold. If your instrument allows you to set an acquisition gate, you can get the answer you're looking for by lowering the threshold to include all of the events you want to count, and restricting your analysis to events within the gate. "Cellular events" is a tricky phrase; strictly speaking, unless you sorted everything, you couldn't be sure which of the near-threshold events were cells and which were noise, debris, etc. It is now generally appreciated that good gating is essential for good flow cytometry; threshold definition is probably at least as important. In this context, it seems to me that a lot of manufacturers (myself included) might improve their hardware design to permit multiparameter thresholding. The Coulter Elite has this feature; some of my Cytomutts, the B-D FACSCalibur, and the Bio-Rad have two-parameter thresholding, while the rest of the Mutts and B-D's instruments and Cytomation's MoFlo have single-parameter thresholding. As far as I know, nobody makes an instrument with two threshold levels for the same parameter, which could be used to count subthreshold events; this feature was incorporated in the Block instruments 20 years ago, primarily because they were originally designed as blood cell counters. -Howard
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