Nathalie, For murine dendritic cells, I would recommend a combination of Class II, CD11c, CD11b, , CD19, and CD8a. Without CD8a, you will be unable to differentiate the myeloid and lymphoid DC populations in the spleen. For bone marrow derived DCs, this is not an issue. In the mouse, most B cells constuitively express high levels of class II MHC, and thus cloud somewhat the DC population in a plot of class II versus CD11c. I recommend running CD19 or B220 in the tube to eliminate these B cells from the plot of class II versus CD11c. For peritoneal macrophages, F4/80 is what you want. It is expressed at extremely high levels on peritoneal macs, though be wary of its expression levels in other tissues (splenic macrophages do not have high amounts of F4/80). You can purchase a very nice FITC conjugate of this from Serotec. And a word about DC isolation. I would generally recommend doing no purification prior to running your cells through the sorter. Sucrose based gradients such as Percoll have the potential for changing the phenotype and function of the DC populations, based on the various carbohydrate receptors on the surface of the cells (for example, mannose receptors). You can read about the effects of various isolation compounds on DCs in papers by Hemo Drexhage (for example; J Endocrinol 1994 Mar;140(3):503-12 Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6. Mooij P, Simons PJ, de Haan-Meulman M, de Wit HJ, Drexhage HA.) At worst, I'd recommend a negative selection column. I have used the DC isolation cocktails and columns from StemCell Technologies with great success. Using sucrose gradients and adherance purifictation is asking for trouble. It takes us about 20 minutes to run the entire cellular content of a murine spleen (about 100 x 10^6 cells) through a sorter, and keeping the cells at 4 degrees during sorting and collection, we feel we've done as little as possible to affect the function of these cells. If you need help preparing your cell suspension for those types of speeds, just drop me a line and I'd be glad to help. Good luck Kb -- Keith Bahjat, Ph.D. Applications Engineering Manager Cytomation, Inc. Fort Collins, Colorado keithb@cytomation.com www.cytomation.com On 1/10/02 7:58 AM, "Nathalie Tessier" <tessien@ircm.qc.ca> wrote: > > Happy new year to all of you, > > One of my users is having a request and I was wondering if some of you could > have some advices on mouse dendritic cells staining. I know that we can use > the > CD208 for human cells but was wondering if there was a more specific marker > for > mouse. > > Thanks in advance > > Nathalie Tessier > Cytometrie en flux > Institut de recherche cliniques de Montreal > > Pavel Chrobak wrote: > >> Dear Nathalie: >> We would like to do sorting for mouse spleen and BM derived dendritic >> cells, as well as for peritoneal macrophages. Currently we are using Percoll >> followed by o/n incubation and then staining with CD11c FITC for DCs and o/n >> adherence on plastic followed by staining with Mac1 PE for macrophages. We >> were wondering if this is sufficient, or whether we should use different >> staining. >> Thanks. >> Pavel Chrobak >> Laboratory of Molecular Biology >> email:chrobakp@ircm.qc.ca > > -- > Nathalie Tessier, M.Sc. > > Adjointe au directeur de l'administration de la recherche > Responsable, Service de cytométrie en flux > > Assistant director, Administration of research > Head of flow cytometry service > > Institut de Recherches Cliniques de Montréal > 110, ouest avenue des Pins > Montréal, Québec, Canada > H1R 1N7 > tel:(514) 987-5608 > fax:(514) 987-5724 > >
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