Good Morning or Good Evening, depending on which side of the plant you dwell. Following Alice Givan's and others train of thought on this matter of one cell vs an aggregate; pardon my naivety on this, could not this matter be easily settled not by microscope alone, but using the same methods applied to DNA analysis in discerning 2N vs 4N. A hard copy for all non-believers and believers alike. Howard S. Mostowski -----Original Message----- From: Alice.L.Givan [mailto:Alice.L.Givan@dartmouth.edu] Sent: Tuesday, November 06, 2001 7:45 PM To: cyto-inbox Subject: Re: cd4 cd8 coexpression Hello Flowers, I just wanted to re-inforce Ken Ault's comments about artifactual co-expression of CD4 and CD8 due to coincidence of two cells in the laser beam. Two cells can coincide in the beam either because they are physically aggregated into a clump or because they just happen (by statistical probablility) to be suspended in the same volume of sample buffer as it moves past the laser. A coincidence artifact should be suspected if: 1) as Ken said, the frequency of these CD4/CD8 doubles decreases when the flow rate is decreased (although this may not happen if the cells are in actual clumps). 2) the intensity of each color on the double expressors is the same as the intensity of each color on the relevant single expressors. In other words, if the double expressors form the fourth corner of a perfect rectangle on a dot plot (with the negs, the PE+ singles, and the FITC+ singles forming the other three corners), then you should be suspicious. Alice Alice L. Givan Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center Dartmouth Medical School Lebanon, New Hampshire NH 03756 tel 603-650-7661 fax 603-650-6130 givan@dartmouth.edu
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