Sorting issues

Hello to All,

I have been confronted with some sorting problems and some spurious signals on my MoFlo
lately, and was hoping someone could provide some insights from personal experience.

1.  The first problem is a sort in which I am trying to sort out 4 different populations
on a PE/APC plot, also using a lineage negative window, and different levels of GFP
expression as sorting parameters.  I usually get pretty clean GFP sorting, when I check
the sorted samples, sometimes the PE/APC windows are clean, although sometimes there is
obvious contamination from another sort window or completely outside the desired window,
and usually the lineage negative window is enriched, but rarely more than 70% pure, and
this does not improve with a second sort.

2.  The second problem I had recently was an experiment comparing PE/APC to PE-Cy7/APC to
define a certain double positive population.  After checking all the single color
compensation controls(including a FITC only control), and seeing signal only in the
appropriate detector for each color, when I put on the actual samples I saw a very strong
signal appearing in the FL1 detector.  This did not occur when repeated on a different
cytometer.


Cytomation recommended replacement of the MFIO board, but a new board does not function
properly in  my MoFlo (it will not allow sorting on compensated parameters).  They are
trying to ascertain if there are software issues causing the board not to work, but they
have not isolated the problem(s).  I suspect there is a problem with the compensation
circuitry.

If anyone has had  similar problems, or has any insights to offer, I would greatly
appreciate hearing from you.

Thanks.

Joanne 


Joanne Yetz-Aldape
Manager-Flow Cytometry Core Facility
Cutaneous Biology Research Center
MGH-East; Building 149
13th Street
Charlestown, MA 02129

Phone: 617-724-0247
FAX: 617-726-4453
E-mail: joanne.yetz-aldape@cbrc2.mgh.harvard.edu