>Basically, you need to run at high flow rates, of >50,000 cells/s and then
>you need to threshold out most RBCs (say 90%, when 0.1% are WBCs), but the
>remainder comprise WBCs at or below 1/1000 cells. Every white cell has 1, 2
>, 3, 4,.. RBCs coincident except for a MoFlo MLS system with 5 microsecond
>dead-times, where you get mainly none, one or two coincident RBCs.
>
>My patent protocols generally use a pulse width and a DNA dye to add an
>exclusion gate for non-nucleated cells, but then you must resolve the issue
>of RBC coincidences and how they affect gates for the WBC subsets and the
>dispersion in fluorescences of positives and negatives!
>
>If you don't have a MoFlo, then buy one; else forget it!
>
FYI,
MoFlo is *not* the only commercial sysytem that is high speed with a deadtime
adjustable down to approx. 5 microseconds. We often run with high throughputs on a
BDIS Vantage + TurboSort using deadtimes at ~ 5 microseconds.
I believe the losses, etc., of the MoFlo are very similar to the modified Vantage
equipped with the TurboSort option. So.... I don't think we'll forget it.
We have also used benchtop instruments (FACScan, FACS Calibur, etc.) triggering on
CD3 immunofluorescence (as Howard described) with good results.
Joe Trotter
The Scripps Research Institute
JT