Hi, I would like to thank everyone who replied with helpful suggestions. I understand that 1: 100 million is crazy- I always did. I would also like to elaborate more at this time about the experiment(s). The cells that will be sorted are your basic cell line- some are adherent and some are suspension. All are basically the same size. The cells will be used to screen a cDNA library for certain antigens, which are either tagged with a reporter like GFP for or labeled by some manner for surface expressed antigens. However we are talking about one antigen for each experiment. I don't see anyway for multiple labels to help pull out positive events. I have detected 1:1 million cells in the past with analysis (not sorting) on a Coulter Elite looking at bone marrow or stem cell products by using many markers and 6 parameters but in this case I don't see how it is possible. The cells are viable during the sort but do not need to be afterward- we just need to be able to recover the DNA. I thought the pre-enrichment for one of the experiments might work but after talking to one of the researchers here- it is not. It is this experiment that could have 1:10 to 100 million positive events. We are working now to try to come up with something more attainable for the sort. There are several other experiments that may be looking a 1:100,000 or 1: 1 million positive events. The other sorts I will be doing are easy. These are the challenging ones. I will try some of these suggestions and determine what I can detect with some trial experiments. Additional suggestions will always be welcome. Thank, Todd -----Original Message----- From: Ann Atzberger [mailto:ann.atzberger@EMBL-Heidelberg.DE] Sent: Wednesday, August 14, 2002 12:40 PM To: cyto-inbox Cc: cytometry@flowcyt.cyto.purdue.edu Subject: Re: sorting rare events- 1 in a million or more Hi Todd, you haven't really given any info regarding what kind of cells you need to sort. As mentioned in previous posts the best option is to pre-enrich if possible. If not possible you need a strategy: one of the things I do for rare cell sorting is to put as wide a gate as possible across the area where the target cells are expected to fall and include 5% to 10% of the neg population. This is simply to recover as much of the target cells as possible and also to acquire enough cell to keep them happy in culture. The cells usually go back into culture and are resorted for purity a few days later. If your cells are not going into culture you could if you have enough rerun them through the sorter again to purify. Just how pure do the cells need to be? you do not always need 99% purity- depends on the application. A bit more info if possible would help. Do some trial runs by diluting samples 1:100, 1:1000 etc see how far you can go and tell us. regards Ann At 10:23 12.08.02 -0400, you 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 >
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