Hello Cliff, Peter, and others, In response to Peter's reference, I presented a poster at ISAC in 1998 where mouse eosinophils were sorted at 35 psi and event rates ranging from 10-20,000/second on the Vantage w/TSO and then measured for changes associated with apoptosis or otherwise increase in cell death. The sorted cells showed little difference in physiological characteristics as measured by mitochondria membrane potential and annexin v staining when compared to unsorted eosinophils. With the help of Rich Konz and CompuCyte Corp. we were able to measure these changes on the Laser Scanning Cytometer (LSC) and then visualize the morphology of the cell populations under the microscope. After sorting we then were able to show eosinophil chemotaxis to eotaxin in both populations (unpublished observation). Since 1998 I've done many high pressure sorts ranging upwards of 45psi (house air doesn't go higher than 55psi). We've sorted many types of primary cells from human and mouse as well as cultures, extracted RNA from these cells, measured calcium responses, as well as the above, with no ill effects versus unsorted/unmanipulated cells. This work has been done with a Vantage w/TSO and a Vantage SE. Regardless of instrumentation, in my opinion most cells sorted at high speed and high pressures, at least up to 45psi (my experience), behave normally. I've been debating getting a N2 tank and running higher pressures but haven't gotten around to it. I know that many of you are accustomed to running at significantly higher pressures and I'm sure the rest of us would be interested in knowing just how high can you go and still obtain viable and functional cells. These concerns and questions are worth answering. A cadre of flow jocks in the Boston Area led by Allen Parmelee from Tufts University meet every first Monday of each month to discuss these issues and others related to high speed sorting. If anyone in the area is interested in attending these meetings, I'd suggest contacting Allen. I have his email if you are interested, let me know. Happy Sorting, Chris Chris Groves Millennium Pharmaceuticals Inc. 75 Sidney Street Cambridge MA 02139 617-679-7495 groves@mpi.com Peter Lopez wrote: > > Hello Cliff, > We have looked at a number of cell types to try and > address these questions on the MoFlo. Others have examined this also using > different > instrumentation. We had a poster on viability at ISAC in 1998 (Cytometry > Suppl 9, page 122 ), and will have > a follow-up poster at ISAC in May. Initially we looked at viability by PI > after sorting, as well as performance > in cell culture. > We have not yet looked at the more specific functional > characteristics that you mentioned, > although I think others have... (see Chris Groves, Cytometry Suppl 9, page > 137). > > I don't think it's too late to try and organize an informal get > together at ISAC for those > interested in sharing their experience with cell "quality of viability" > after high speed sorting. Please let me > know if you're interested. I know Dave Coder and Ger van den Engh will have > a tutorial on > high speed sorting at ISAC, and may also address these issues. > > One other resource for you to try is the MoFlo Users Group, since > there you will find folks who have > been doing this type of thing for quite some time, and they may have more > specific information > related to the cells you're using. > > Peter > > > ---------- > > From: Cliff McArthur[SMTP:cytocliff@netscape.net] > > Sent: Monday, March 06, 2000 10:16 AM > > To: Cytometry Mailing List > > Subject: HOW viable after high-speed sorting? > > > > > > Hi there, flow aficionados. > > > > I know we've all addressed the post-sort viability (after high-speed > > sorting) > > question many times over. What I am looking for is not "whether" but "how > > much," so to speak. To that end, I'd like to ask the List if anyone knows > > of > > any published work that has addressed the question not of whether or not > > cells > > are viable after such sorting (we know they are, or can be) but "how" > > viable, > > that is, do they produce less cytokine or other product, proliferate a > > little > > more slowly, or survive for less time, etc., etc. under the same > > conditions > > versus "low-speed" sorted cells? Shucks, references that just address > > this > > question, if it is not the primary objective of the work, would be great > > to > > have. > > > > If anyone is willing to share their unpublished insights, I'd love to hear > > them as well. > > > > Thank you very much, > > Cliff McArthur > > University of California at San Francisco > > Departments of Medicine and Immunology/Microbiology > > 415-502-6860 > > > > ____________________________________________________________________ > > Get your own FREE, personal Netscape WebMail account today at > > http://webmail.netscape.com. > >
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