>Hi, > >I have a rather simple sounding question, particularly for the sorters >out there. I am looking to positively separate CD4, CD8, and monocytes >from Ficoll-hypaque separated blood samples. I intend to use avidin >columns and biotinylated antibodies. Obviously CD4 and CD8 will be use >for the isolation of these lymphocytes, but the best monocyted marker is >less clear. I have considered CD14, CD15, and CD33. My goal is to >obtain a pure monocyte sample, not necessarily the entire monocyte >population. The cells will be fixed immediately after they are >isolated and the subsequent manipulations are not affected by the >activation state of the cells. Does anyone have experience with these >surface markers or others that would be more appropriate for isolating a >relatively pure monocyte population? > >Thanks in advance > >Wallace Lauzon >Dept. of Microbiology and Immunology >University of Ottawa 100% of monocytes stain brightly with anti CD11c. Sigma sells a cy5-pe conjugate, very good, or any of the PE conjugates are good. Some lymphocytes do stain dim with CD11c, however, but discrimination on a sorter is not a problem. Discrimination on a column would be a problem. There are no specific markers for myelomonocytic cells analagous to lymphoid cells. That is, though CD19 is B cell specific and CD3 is T cell specific, CD14 eis expressed on neutrophils as well as monocytes, although at lower levels. True for CD11c. Some markers, very bright, such as CD13, are expressed on same levels on neutrophils and monocytes. If you are looking for specificity, your best bets are CD14 or CD11c, both of which are very bright, and can discriminate between the dull expressers, neutrophils and less mature monocytes in the case of CD14, some lymphocytes and neutrophils in the case of CD11c. The last time I looked, CD33 wasn't that great, because it wasn't very bright. CD15 is not best for monocytes becasue of the wide heterogeneity of expression, and is much better for neutrophils, which are extremely bright. ron Ronald L. Rabin, M.D. Clinical Associate Cytokine Biology Unit LCI/NIAID NIH phone: (301) 402-4910 fax: (301) 496-7383 e-mail: rr84g@nih.gov
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