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. > >
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:11 EST