>Hello everyone; > >I have a colleague who wants to isolate murine dendritic cells by flow. We >know that there are no commercially available markers for these cells. Some >people are using I-A, size and exclusion of B cells as a way to collect cell >populations enriched for dendritic cells. could someone please provide me >with some more details concerning this approach or any other that may be >useful. Any information is much appreciated. Thanks in advance. > > > >Edward F. Srour, Ph.D. >Associate Professor of Medicine and Pediatrics >Indiana University School of Medicine >1044 West Walnut Street, R4-202 >Indianapolis, IN 46202-5121 >phone: 317-274-3589 >fax: 317-274-0396 >email: esrour@iupui.edu By murine do you mean rat or mouse (aren't both "murine"?)... If you mean mouse then the following papers (and others) from Ken Shortman's group at WEHI might be helpful (I can't remember the exact paper which has the best description of the method), Kronin, V., D. Vremec, et al. (1997). "Are CD8+ dendritic cells (DC) veto cells? The role of CD8 on DC in DC development and in the regulation of CD4 and CD8 T cell responses." International Immunology 9(7): 1061-4. Kronin, V., K. Winkel, et al. (1996). "A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production." Journal of Immunology 157(9): 3819-27. Süss, G. and K. Shortman (1996). "A subclass of dendritic cells kills CD4 T cells via fas/fas-ligand-induced apoptosis." Journal of Experimental Medicine 183(April 1996): 1789-1796. Vremec, D., M. Zorbas, et al. (1992). "The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells." The Journal of Experimental Medicine 176: 47-58. Wu, L., D. Vremec, et al. (1995). "Mouse thymic dendritic cells: kinetics of development and changes in surface markers during maturation." European Journal of Immunology 25: 418-425. There are other protocols but they seem to selectively loose some of the sub-populations (as can these protocols from Ken - at a recent meeting I heard him say that they now leave CD4 out of their negative selection cocktail as some splenic DCs can express it, or a least have it on their surface (they may have picked it up from T cells)). We have modified this protocol to the following... * Collagenase digest of spleen, with EDTA present for 5min at the end to help dissociation of Tcell/DC rosettes * Nycodenz density separation for light cells * Postive selection for CD11c (antibody clone N418) on a MACS column. We use anti-FITC beads for these but direct anti-CD11c beads are available. We find that this step (or the negative selection step) necessary in order to be able to sort a reasonable number of DCs in a reasonable time. * Sort for B220- and N418+ cells which we separate into CD8+ and CD8- fractions See the following paper... Smith, A. L. and B. Fazekas de St. Groth (1999). "Antigen-pulsed CD8a+ Dendritic Cells Generate an Immune Response after Subcutaneous Injection without Homing to the Draining Lymph Nodes." Journal of Experimental Medicine 189(3): 593-598. Let me know if you want more details (I am bit busy to write them at the moment, maybe after Easter) Adrian Smith ****************************************************** Adrian Smith (PhD Student) T CELL BIOLOGY GROUP Centenary Institute of Cancer Medicine & Cell Biology Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. Ph: 61-2-9565-6197 Fax: 61-2-9565-6103 A.Smith@centenary.usyd.edu.au OR adriansmith@email.com ******************************************************
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