PHA action

vjovic@hermes.beotel.yu 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) 
Oh, yes
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
             Viktor


BIG THANKS TO EVERYBODY ON REPLYS FOR " K562 MARKER" QUESTION


Dr Viktor Jovic
Experimental Laboratory
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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