Your problem may be the CD8 Cy-chrome. This conjugate is known to bind "nonspecifically" (through a CY-chrome receptor) to cells of monocyte lineage and cannot be blocked by neutralizing the Fc receptors. I know because I've tried. Call the manufacturer. I have it from a reliable source that this conjugate has been reworked to eliminate this problem. Margaret >Hi Marcos, I use 10% human AB serum to incubate the cells for 20 minutes at >4 degrees, then wash and pellet the cells. Afterward you can use your >antibodies. I hope it will help. Regards Rafael >>Hi colleagues, >> >>I tried to browse the archive to find the answer to this question, which I >>think might have been addressed before, but without success. >>Does anyone have a protocol for fc-receptor blocking with human IgG when >>staining ficoll-separated PBMCs? I usually work with 0.5 to 1 x 10 to the 6 >>cells per tube. My routine conjugates are CD3-APC, CD4Pe or FITC and >>CD8Cy-chrome. >>Thank you, >> >>Marcos da Silva >>Pediatric Immunology lab - UMass Medical School >>Marcos.DaSilva@umassmed.edu > > > \|/ > (o o) >________________________________oOo__(_)__oOo______________________________ ___ > ___/\_ | Rafael Nunez mailto:rafaeln@vetvir.unizh.ch > / o \/| | University Inst.for Virology http://www.vetvir.unizh.ch/ > / _| | Winterthurerstr. 266a Telephone: (+41) 1 6358710 > /_/\__/-\/ | 8057 Zurich SWITZERLAND Faximile : (+41) 1 6358911 >___________________________________________________________________________ ___ "The opinions stated above are the authors own, and should not be construed as U.S. Government endorsement of a specific vendor, nor used in any publication to indicate endorsement by the U. S. government or any of its agencies." >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Margaret M. Tropea Critical Care Medicine Department National Institutes of Health (ph) 301-496-7752 (fax) 301-480-3389 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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