Here it is: http://www.innovativecelltech.com/accutase.html from Phoenix Flow in San Diego. Dave ======================================== David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com or dcoder1@hotmail.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Ress, S, Stan, Dr" <sress@uctgsh1.uct.ac.za> To: cyto-inbox Sent: Monday, May 20, 2002 4:58 AM Subject: Re: EVC 304 abort rate > Hi David, > > Please could you look up details on the magic Elixer that gets > adherent cells off in great shape, once you're on 'terra firma', and > post to discussion list, or else reply to me, it sounds like > something I could really use!! > > Many thanks, > > Stan > > > > > Reply-to: "David Coder" <d_coder@MSN.com> > > From: "David Coder" <d_coder@MSN.com> > > To: Cytometry Mailing List <> > > Subject: Re: EVC 304 abort rate > > Date: Wed, 15 May 2002 08:38:56 -0700 > > > > > Very useful comments on sorting improvements. I agree that a major problem > > is the debris level. Starting earlier in the process of getting adherent > > cells into suspension would be using the magic elixir that Phoenix Flow > > Systems sells (sorry, Kevin, I can't remember the name and I'm too cheap to > > plug into the Airphone here at 35,000ft and pull up your webpage). Not only > > do you get a much cleaner cell prep, but you'll have a higher yield of > > viable cells (less cell damage, less debris). Secondly, you may also > > benefit from using a nozzle with a larger diameter. If the cell is occupying > > too much of the stream as it exits the nozzle, stream perturbations result. > > Also, I'm guessing here, but a larger cell in a smaller drop and drops alone > > may charge differently as they leave the stream thus giving rise to ugly > > sort streams. Granted, as you go up in nozzle diameter, sort rates will > > drop, but there's probably an optimal set of conditions where sort rate and > > abort are best. > > > > Dave > > ======================================== > > David M. Coder, Ph.D. > > Consultant in Cytometry > > email: d_coder@msn.com or dcoder1@hotmail.com > > tel./messages: 206-499-3446 > > ----- Original Message ----- > > From: "Mostowski, Howard S." <mostowski@cber.fda.gov> > > To: cyto-inbox > > Sent: Monday, May 13, 2002 11:34 AM > > Subject: RE: EVC 304 abort rate > > > > > > > > > > David, > > > > > > The problem is not you, but the cells themselves, that is not > > > exactly accurate neither. The cell are in a messy "FACS" media. This > > > usually happens with any of the adherent cell and specially with cells > > that > > > are fixed. I too have seen this problem appear, but it is around 50% at > > 10 > > > to 12k cells/sec at a sheath pressure of 14. You probably noticed two > > other > > > facts [pardon the pun], but if you reduce the your sample pressure thus > > > reducing the number of cells/sec your instrument has to make a decision, > > the > > > abort rate drops dramatically. You did not mention if you had the > > Accu-drop > > > attachment, but if so you would notice that it is virtually impossible to > > > hold your side streams perfect even though they where with the control > > > beads. I use a two droplet-delay setting. > > > Great now you know at least two of us has seen this phenomenon; that > > > does not explain what is happening and what I can do to somewhat alleviate > > > it. So here goes. > > > 1. Filtering is a must as it will remove a great deal of the > > > artifacts cast off from the damaged cells as well as un-dissolved > > > preservative. You probably will not get rid of all the cellular debris as > > > more garbage is constantly being produce by the damaged cells sitting in > > > your sample tubes. You might think you could reduce the problem by > > > increasing the threshold [I keep my FCS threshold at 20], it will not > > work. > > > The reason is, even though the artifacts are out of the picture > > > electronically they are still there physically. Because they are still in > > > the equation, your instrument has to make a decision about cell + debris > > and > > > it is not in your favor. > > > 2. The other way to reduce the abort rate is as mentioned above is > > > to reduce your sample pressure to below 3000 cells/sec. > > > 3. The third way is to increase the concentration of cells. By > > > increasing the concentration of cell incorporated with a reduction of > > > cell/sec allows you to have a sample stream of a smaller diameter; thus > > > reducing the chance of debris coming past you beam at the same time, but > > > here you have to have a feather touch on your on your sample differential > > > knob. > > > I hope this helps somewhat, Cheers and good luck. If any ones else > > > has some other techniques, please put it in the open format, because I am > > > always ready to learn frame someone more wiser. > > > > > > Howard > > > -----Original Message----- > > > From: Corry, David [mailto:David.Corry@uwe.ac.uk] > > > Sent: Monday, May 13, 2002 7:23 AM > > > To: cyto-inbox > > > Subject: EVC 304 abort rate > > > > > > > > > > > > Hi all, > > > > > > I've recently started analysing ECV304 endothelial cells on my FACSVantage > > > and > > > have been having problems with a high co-incidence abort rate - typically > > > 75%+!! > > > The cells are fixed with 1% paraformaldehyde/1% FBS in PBS and stained > > with > > > FITC or RPE conjugates. The problem doesn't seem to be as bad with unfixed > > > cells or with some of the other adherent cell lines I use e.g. C20/A4 > > > chondrocytes. > > > Could it be a problem with the fixing protocol? Anyone got any ideas or > > > suggestions? > > > > > > Thanks in advance > > > > > > Dave > > > > > > ---------------------------------------- > > > David Corry > > > Centre for Research in Biomedicine > > > Faculty of Applied Sciences > > > University of the West of England > > > 0117 344 3397 > > > Email: David.Corry@uwe.ac.uk > > > > > > > > > > > > > > > > > > >
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