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Now we set quads in the #2,3,4 histogram and compensate all
permutations.
We keep compensating the "single positive" populations (e.g. in hist #4,
CD8+CD4neg) until the Median fluorecscence of this population on
the axis of the negative parameter (i.e. CD4 in this example) is the same
as that of the double negative population (i.e. CD8neg,C4neg).
This strategy works very well because you are compensating in
the fluorescence range of your actual populations (which may or may
not be the case with beads). However if your experimental tubes contain
combinations of MABs which stain very brightly (CD38, CD45RA...)
you may have to overcompensate the CD4/8/3 tube in order to keep the
brightly staining populations "orthogonal." In either case, the use
of a standard MAB combination with known populations patterns
provides a frame of reference for multicolor compensation and is very
cheap.
The new software for the Coulter XL makes compensation very easy
because you can "drop and drag" populations to change settings and
all parameters can be compensated against each other (e.g. Fl3-Fl1).
Have fun compensating, call me if you have any questions. (do not
DECOMPENSATE!)
Vera 412-624-9599
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web