Ann, I second your support of Jim's suggestion that we re-name this as "high pressure sorting," thereby indicating that we're doing more than merely going really really fast. MAK. Ann Atzberger wrote: > As a rule I run most of my cell types at 40psi-70um nozzle, big cells > 30psi-100um nozzle, and on occassion yeast and bacteria at > 60psi-50umnozzle. Most of the cells go back into culture: when I worked > with the MoFlo demo instrument cell sorting was done paralell to the FACS > Vantage and viability etc, compared. The users did not report any changes > in viability and so far it's not an issue. Viability applying to sorted > cells that are still proliferating a week after sorting: -but a lot of > things affect viability after sorting e.g if only a small number of cells > has been sorted they may not feel too happy if too dilute in culture. Maybe > someone should provide guidelines on how to treat cells after sorting; as I > think quite a few mistakes are made here. > > However; it did not occur to me to see if the fuctionality of the sorted > cells was in any way influenced by the high pressures: I will now pay a bit > more attention to this. I think I read here on the list or maybe somewhere > else that it's a good idea to put the sorted cells back into their optimum > environment to let them recover from sorting before continuing to do any > assays with them. > > As for high speeds; well this always depends on the concentration of cells > one gets and more often than not you have high pressure and not-so-high > analysis rates.This depending on the sample volume you feel comfortable > working with. Adherent cells can get very sticky if too concentrated. I > don't increase the sample differential above sheath pressure to speed > things up, as this seems to affect the side streams. However, yeasts and > bacteria I can run at fantastic speeds but I don't get them very often. > > So as Jim says it's mostly high pressure with various analysis rates: so > I'd be quite happy to use the term high-pressure sorting. > > Regards > Ann > > At 10:01 07.03.00 -0600, you wrote: > > > >IN pertaining to this e-mail and many like it, I was wondering if there is a > >possible way to re-define the phrase "High-Speed Sorting". > >I tend to visualize this phrase to refer to events/sec as being analyzed by > >the instrument. In some cases it could be >10k/sec in some >30k/sec. All > >depending upon the operator. > > > >Viability comes into question in all aspects of sorting, whether High or > >Low. The factor that governs this is not the speed but the "pressure" at > >which sorting occurs. I can sort at 12k/sec on my Vantage at 30 psi up to > >45 psi. I have even sorted at 12k/sec on the old FacStar at 15 PSI. > >Certain cell types do not do well at higher pressure greater than 25 psi > >some do well even at 60 PSI. > > > >When it comes to viability, is this immediate after sorting viable cells? > >Cells viable 4 hrs post sorting or even 24hr post sorting? Some cells may > >loose their potential to proliferate/respond at higher pressures. This is > >only seen after days of culture. > > > >In all. You have to take into account the sorting pressure and not the > >speed to evaluate the sorting effects on the cells. > > > >I suggest we refer to sorting as "high pressure sorting" vrs. "high speed > >sorting". IN doing so it would be wise to relate what pressure we are > >sorting at and what type of cell we are sorting. > > > >Many are doing the "High Speed Sorting", but I would bet that we are all > >using different pressures to achieve this. > > > >Jim Houston > >Coordinator, Flow Cytometry and Sorting > >Cell and Gene Therapy Program > >St. Jude Children's Research Hospital > >Memphis, TN 38105 > > > >ph:901-495-2926 > > > > > > > > > >-----Original Message----- > >From: Cliff McArthur [mailto:cytocliff@netscape.net] > >Sent: Monday, March 06, 2000 11:17 AM > >To: cyto-inbox > >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. > > > > -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry <http://www.cancer.med.umich.edu/flow_cytometry> phone: 734-647-3216 fax: 734-936-7376 kukuru@umich.edu
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:11 EST