 |
 |
 |
 |
When setting up for quadrant analysis, people frequently comp until the FL2 top
of the FL1 stained population is the same as the top of the double-unstained
population - and then set quadrants on the unstained. This leaves the center of
the comped population lower than the unstained (a potential definition of
overcomped). If you get a population that is brighter in FL1 than you set up on
the center is not low enough to keep the top edge below the quadrant marker (not
overcomped enough). For this comp and analysis strategy to work you need to set
up comp on the brightest thing you are likely to get.
For non-saturating dye situations, the fewer targets the more dye per target.
Thus samples with low retics might have brighter ones. Leukopenic samples with
fewer nucleated cells to take up dye might also do this. So Ray, are the
samples with the artifactual FL2 ones where the FL1 is brightest?
The issue has now been expounded upon, I'm sure this is not news to most of you.
Raymond B. Hester wrote:
> doing 2-color FACS (we have a 440) with thiazole orange (TO) to
> detect retics and a phycoerythrin conjugated antibody to look at another
marker
. The problem we are having is that
> sometimes the TO-only stained cells sometimes appear in the lower
right (530/30) quadrant as expected but sometimes they appear in the
two-color quadrant as if they were doubly stained.
Ray: The problem you describe is actually predictable based upon the
spectral properties of thiazole orange (see Lee et al, Cytometry 7:508,
1986 or editions of Howard Shapiro's excellent text). TO emits further
into the spectral range of PE relative to FITC. If you need to do two
color work with TO, you will need to use a Moab signal in the red range,
such as tri-color or PerCP.
Bruce H. Davis, M.D.
****************************
Director of Analytical Cytometry
Dept. of Clinical Pathology
William Beaumont Hospital
3601 W. 13 Mile Rd.
Royal Oak, Michigan 48073-6769
810-551-5137
FAX 810-551-8057
|
|
|
|