Dr Lawry, We have sorted and characterized both DC's from skin and the so called macrophage "derived" DC's. The most striking characteristic in both of these cell types is the surface expression of HLA-DR. In which the DR expression on skin DC's is much brighter than the M0 derived DC's. Depending on whether the DC's are conjugated with T-Cells or not will define the physical characteristics. Generally, these cells scatter high 90 and mid-scale using peak forward scatter, however, the skin DC's are larger and may require tuning down the 90 scatter. With all this in mind I recommend staining with the following combinations; DR/14-56-20/CD3 : Background staining for the monocyte derived DC's. As cells "transform" they should loose the CD14 marker and none should be positive for 20 or 56. If this is done correctly all of these could be PE because you really do not need these percentages. This is our combination for sorting. DR/80/CD3 : On DC's (unconjugated) they will be positive for DR and 80. DR/83/CD3 : On DC's (unconjugated) they will be positive for DR and 83. Stephen P. Perfetto, MS.,MT. (ASCP) Walter Reed Army Institute of Research Department of Molecular Diagnostics and Pathogenesis 1600 East Gude Drive Rockville, MD. 20850 _______________________________________________________________________________ Subject: Dendritic cells From: J.Lawry@sheffield.ac.uk at Internet_Gateway Date: 1/27/98 9:35 AM Dear All, We are trying to characterise dendritic cells in stimulated cultures of PBMCs. We have a panel of potential markers of dendritic cells but are having problems finding their characteristic scatter patterns for gating purposes. Back gating gives no clear area. Can anyone help? Where do these cells lie on a normal FWD vs SSC plot of "whole blood" - in the LGL/monocyte area, or towards the neutrophils? Many thanks, John. Dr John Lawry, Institute for Cancer Studies, University Medical School, Beech Hill Road, Sheffield. S10 2RX. Tel:0114-271-3023 (lab), 0114-271-3774 (office) Fax:0114-271-3515 Email: J.Lawry@sheffield.ac.uk Web page: http://www.shef.ac.uk/uni/academic/A-C/csi/Staff/John/JL.html
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