Hi Andy, Question about the CD3 FITC control - do you normally see that profile of three populations? Your "corrected" two-colour picture does not show that. I have seen something like this, apparently caused by abnormally high concentration of one of the reagents. This can happen if the reagent is not properly re-suspended. It can also be a mistake in dilution, easy to do after a holiday break. HTH, Joseph. At 04:06 3/1/2002, Andrew Oberyszyn wrote: >Hi Flow people! >First let me wish everybody Happy Holidays and keep up the great work on >this newsgroup! > >I ran some 3 color samples (CD3 FITC/CD4 PE/CD8 PE-Cy5) on my Beckman >Coulter Elite today and I saw something that didn't make sense to me. I've >attached three slides of the histograms that I got. > >I ran the single color positives and compensated so that the mean values >where correct (see Slide 1, compensation settings #1). I then ran the >samples (example of one on slide 2). There where 2 populations that didn't >seem right to me (looked like they where undercompensated). I increased >the compensation so that it "looked right". I then ran the single color >controls again using the increased compensation values (slide 3). The mean >values I got seemed to be overcompensated. > >I apologize for being somewhat ambiguous in the descriptions above (I'm >trying to at least get out of here on time today!) but the histograms in >the attachment are more informative. > >I've done three color work before but have never come across this >problem. The user says that the antibodies are the same (i.e. not >different lots within an antibody throughout the samples). > >I realize that alot of people are on vacation already and responses to this >might be sparse but any help would be greatly appreciated (Mario? Help!) > >Thanks in advance! >Andy -- Joseph Webster, Flow Cytometry Facility Centenary Institute, Sydney, AUSTRALIA. Phone +61-2-9565-6110
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