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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