Fellow Flowers- We have a very strange problem and I'm at a loss to explain. My colleague is staining fresh human LWB with CD3-PERCP and CXCR3-PE. When the CD3-percp control is run on the Facscan, itlooks ok, comps fine & nothing remarkable is noted. When the CD3+CXCR3 is run, it appears that the CXCR3-neg CD3's are GONE. [As in quenched?] In fact, this person was doing a series of CXCR3 Ab dilutions and the more dilute the CXCR3 Ab, the more pronounced the CD3 quench. I reran her samples completely uncompensated-(data posted at this link) http://www.cotleur.com/CotleurMain.data/Components/facsdata.html and you see that on left is uncomp'd CD3 alone, and the right- uncomp'd CD3+CXCR3. ObserveThanks the shift after addition of the CXCR3; so much so that the plot almost appears to actually be compensated. So here's the wrench: 1) this stain/combo was the exact stain (except the dilutions) that they ran many times before, never saw this. The CXCR3 dilutions were done for the first time yesterday, and did not show this CD3 shift. (CXCR3 Ab was serially diluted in PBS/2%NCS/+azide, 0.02%). 2) With each increasing ab dilution, the effect is pronounced. 3) the Facscan was just serviced today. Calibrite beads look perfectly normal, the single stain cells look normal also. 4) CD3 paired with CCR2-PE: does NOT see this. I am convinced it is something in their prep, they are convinced it is the instrument (only because their experiment "worked" yesterday, and did not today- post instrumet service). Has anyone EVER seen anythiung remotely like this? All comments welcome! Bunny ******************************************************* Bunny Cotleur +*+ Bunny Cotleur Cleveland Clinic Foundation *+* 2001 Lester RD Neurosciences NC30 +*+ Valley City, OH 44280 9500 Euclid Avenue *+* 330-483-4800 Cleveland, OH 44195 +*+ bunny@cotleur.com 216-444-1164 *+* cotleua@ccf.org +*+ ******************************************************* When you do something, you should burn yourself completely, like a good bonfire, leaving no trace of yourself. (Shunryu Suzuki)
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