I'll be interested in the responses you get here but suggest you may also want to try magnetic separation of teh rare cells if you have a good and selective antibody to the specific target. This apparatus, developed at Immunicon, is particularly interesting for simultaneous separation and visualization of rare cells in a sample in a microscope. Flow chart for the magnetic separation and analysis of a cell suspension. Cells are treated with an antibody or a biotinylated or DSB-X biotin–labeled probe that binds to cell-surface markers. The treated cells are incubated with the appropriate Captivate ferrofluid conjugates, which bind to target cells. The mixture is then transferred to a chamber that is inserted into a magnetic yoke. Under the influence of a strong magnetic field, the cells bound to Captivate ferrofluid conjugates are rapidly separated from the unbound cells. The separate cell populations can be analyzed by both fluorometry and fluorescence microscopy. http://www.probes.com/handbook/print/0703.html [Image] propidium iodide could be added to the sample to detect dead cells that may be magnetically separated. cells can be released by removing them from the magnetic field. Todd Belanger wrote: > Hi, > > I am new to sorting (but I have ten years of flow experience) and we just > purchased a FACSVantage/DiVa. Some of our projects require sorting rare > cells at levels of 1 in a million or ten million. Some of the researchers > say it could be one in 100 million (which seems quite impossible to me). > Does anyone have any pointers or particularly good papers that would help me > in this task? How low can you go (in terms of rare events) and still be > relatively confident in what you sorted? Currently the researchers I will be > doing the sort for has two markers- PI to discriminate live cells and a FITC > conjugated marker. I know more markers would be better for discriminating > rare events but their doesn't seem to be any for this particular experiment. > > Any and all help would be greatly appreciated. > Todd > > Todd J. Belanger > Lab Manager > Cellular Immunology > Vaccinex, Inc. > 1895 Mt. Hope Ave. > Rochester, NY 14620 > email: tbelanger@vaccinex.com > www.vaccinex.com > > ------------------------------------------------------------------------ > Name: winmail.dat > winmail.dat Type: application/ms-tnef > Encoding: base64
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