vjovic at hermes.beotel.yu
Thu Aug 6 17:04:22 EST 1998
Hello, flowers all over
I have a problem with peripheral blood T-lymhocytes subsets
evaluation following stimulation with PHA (10 micrograms per ml
in RPMI + 10% FCS for 96 hours).
What's happening, actually?
FSC/SSC are changing due to appearance of lymphoblasts (which was
expectable) and isotype controls also (they look like a tail
which was also expectable due to increased autofluorescence)
I have noticed brightly stained clusters of " CD3+ CD4+ or CD3+CD8+
population" and dimly stained CD3+ populations for CD4PE or CD8PE in
samples stained with CD3FITC/CD4PE and CD3FITC/CD8PE, both from
Simulset reagents, BD, more continuously distributed than it was
expectable. When quadrant marker was placed according to isotype
controls, the horizontal line of quadmarker passed directly through
the second continuously distributed population, making two
populations of CD4+ or CD8+ T-lymphocytes, briht cluster and dimly
stained tail that continued with no demarkation in CD3+ CD4- or CD8-
area of dot plot.
So, why CD3FITC+ population increases FL-2 fluorescence in dim spectral
area for CD4PE and CD8PE?
I've tried to make adequate monocolor sample controls but marker was
nearly the same as it was when isotype controls were used.
Compensation tool was not succesfull also (FL2 - % FL1, on nearly 48)
The samples were prepared like it was previously described...
bla...bla ( 30 min incubation with mAbs and two PBS- azide washes and
CellFIX addition or not at the end).
Thanks in advance
BIG THANKS TO EVERYBODY ON REPLYS FOR " K562 MARKER" QUESTION
Dr Viktor Jovic
Institute for oncology and radiology of Serbia
11000 Belgrade, Yugoslavia
E-mail: vjovic at beotel.yu
Phone: +381 11 685755 ext.426
Fax: +381 11 685300
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