compensation
Vera Svobodova (svobve@novell1.dept-med.pitt.edu)
Sat, 11 May 1996 14:33:10 EST5EDT
PJJ and others,
We have been doing 3 and 4 color surface/intracellular staining for
several years and this is how we standardized our compensation
when compensation beads were not available:
-we have used lysed whole blood from a normal individual, stained with
CD4 FITC, CD8 PE/RD1, CD3 PerCP/PECy5. The display is a set of 4
2-color histograms:
#1. FSC x SSC for Ly scatter gate based on CD45 x
CD14 tube (CDC purity/recovery guidelines)
#2. CD3 x CD8...you will get 5 major populations (this is a "rough"
definition but it helps for compensation purposes since this staining
yields mutually exclusive and double/triple positive populations and
it is easy to check (i.e. CD8-CD3+ =`CD4+T-cells):
CD3+CD8bright=CD8+ T-cells
CD3+CD8dim (smaller population)=CD8dimT-cells
CD3+CD8neg = CD4+ T-cells
CD3negCD8dim = NK cell subset
CD3neg CD8neg = B-cells, CD8neg NK cells
#3. CD3 x CD4...will yield 4major populations:
CD3+CD4+=CD4+T-cells
CD3+CD4neg=CD8T-cells
CD3negCD4dim="creeping" monocytes into your LY scatter gate
CD3negCD4neg=B-cells, NK cells
#4. CD4 x CD8...gated on LY scatter ONLY will yield 4
MAJOR populations (it can be gated on LY scatter AND CD3+):
CD4+CD8neg=CD4+T-cells
CD4negCD8bright=CD8+T-cells
CD4negCD8dim=CD8dimT-cells and NK cells (changes with LYscatter and
CD3+ gating)
CD4negCD8neg=B-cells and gamma/delta T-cells (changes with LY scatter
and CD3 gating)
All populations listed are ONLY the MAJOR populations, you will see
also some minor (CD4+CD8+...) populations.
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
CD ROM Vol 2 was produced by 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:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
