I am computational guy trying to understand how FACS sorting works in an scRNA-Seq protocol. About sorting single cells in each well of a 384-well plate, I have a question that would be grateful if you could answer it.

The protocol reads that cells are sorted in the wells based on their marker levels. For example it says:

NK single cells were collected from the CD19-/TCR-β- events by gating for NK-1.1+ events in NK-1.1 vs. Gr1.

The part that I don't understand is that how it is possible to know positive/negative events or even to gate on events beforehand? I know that FACS first examines markers and then sorts them into positive, negative or waste collections. Here is my question:

But how do we know what an expression level is acceptable for calling a cell positive, and also in more complicated case, how do we to gate on a subpopulation of cells (like top right quadrant) when no events have yet crossed the laser beam because we don't know the baseline of CD markers' expression levels yet?

For instance, It could be possible that in my panel setup a CD marker can never reach a value more than X, but in your setup it can never reach a value more than X/2.

N.B. As protocol says, subsampling of the main sample for FACS is not possible to infer these values beforehand because it is very likely that the populations of different cell types is not conserved in the subsample.