Hi Seth- Did you add homologous IgG or blocking anti-CD16/CD32 to your samples before you performed the staining with conjugated antibody? Since granulocytes(as well as other leukocyte subsets) have FC-receptors, these FC-receptor bearing cells can stain non-specifically with the FC region of the conjugated antibody that you are using. Here's an example that I found on the web (although with splenocytes rather than whole blood) that illustrates the differences between blocking and non-blocking conditions: http://www.caltag.com/pdf/ApdxK.pdf Hope this helps! Todd At 01:28 PM 8/29/2002 -0400, you wrote: >hi, > >i'm a neophyte at flow cytometry, so i'm hoping someone will be able to >answer these questions. > > >1) i'm interested in monocytes and use CD14. Now, as i understand it >CD14 stains monocytes brightly, and granulocytes dimly. So in all my >samples i've been puzzled to find a large amount of bright CD14 cells in >the region of a standard FSC-SSC plot that is the granulocyte region >(higher SSC then the monocyte region). I still see that classic cluster of >monocytes, but there are also these other cells. > >So we sorted these cells - hi SSC/bright CD14 - and looked at them under >EM and there are plenty of neutrophils there, and monocytes. > >So whats the story with these cells? are there hi-SSC monocytes? why >are these neutrophils bright for CD14? > >2) This isn't really a flow question but maybe someone knows... >We tried to isolate a rare population of CD71/CD14 double positive cells, >but only got 8500 cells from 20 cc of blood. The EM people weren't able to >prepare this little amount for EM. > >So, aside from using tremendous amounts of blood and antibody and flow >time to get several hundred thousand of these cells, Does anyone know of >any EM techniques for preparing small amounts of cells? > > >thanks alot. > >--- >Seth L. Ness M.D., Ph.D >Fellow in Human Genetics >Department of Human Genetics >Mount Sinai School of Medicine > >Phone:212-241-6947 >Fax:212-860-3316 >sln8@columbia.edu > >Ness Gadol Haya Sham Todd A. Johnson Computer Systems Manager UCSD, Department of Psychiatry Dimsdale Lab CTFA 309/ MC 0804 (619) 543-2504 tajohnson@ucsd.edu CONFIDENTIALITY NOTICE: The information contained in this message is legally privileged and confidential information intended only for the use of the individual or entity named above. If the reader of this message is not the intended recipient, or the employee or agent responsible to deliver it to the intended recipient, you are hereby notified that any release, dissemination, distribution, or copying of this communication is strictly prohibited. If you have received this communication in error, please notify the author immediately by replying to this message and delete the original message. Thank you.
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