Hi Andy: What size nozzle are you using and how big are your cells? Are they clumpy, is your abort rate high? If your cells (or cell aggregates) are too big for your nozzle size you can get exactly what you describe. Your nozzle size should be at least 5X the largest particles in your suspension. Even if your nozzle size is appropriate for the cell size, if you have lots of cell aggregates it can create the same type of problem. Try switching to a larger nozzle and see if that doesn't help. Joe Trotter did a great demonstration of this effect at the Bowdoin Course where he had a 70 um nozzle and sorted 5um beads and the streams looked great. He then added some 10 um beads and it still looked pretty good. When he added 15 um beads we started to see some degradation in the streams and when he added the 25 um beads it looked just awful with lots of spray between the center stream and the side stream. It was enough to convince me that nozzle size is important to get a clean sort! I always ask my clients what size their cells are (and if necessary verify what they are telling you with beads of different sizes under a microscope). Also if they have been activated they may in fact be significantly larger. If their samples are traditionally clumpy and the usual remedies don't work I generally will go with a larger nozzle size (helps prevent clogged nozzles as well). Drawbacks are that you have to sort at lower pressures and you have larger drops (i.e. larger sort volume), not to mention you are changing tips and realigning more often as well. But I must say I have seen a considerable improvement in the quality of these sorts in subscribing to this philosophy. THANKS JOE! Give it a try- Regards, Joanne Lannigan, MS Director, Flow Cytometry Core Facility University of Virginia P.O. Box 800734 Charlottesville, VA 22908-0734 Office: 434-924-0274 Lab: 434-243-2695 Fax: 434-982-1071 email: joannelannigan@virginia.edu From: Andrew Oberyszyn <oberyszyn.2@osu.edu> Date: Wed, 16 Oct 2002 16:45:14 -0400 To: cyto-inbox Subject: GFP sorting question Hi FLow-er in Flow land, I've come across an interesting (and confusing) problem which sorting GFP cells. I've noticed several times that I get alot of fanning while sorting. I don't believe it is the Frequency, drive or phase since setting up the sort gives me beautiful side streams and great separation. I first notice this on my Elite and now I have a FACSVantage and 2 of the last 3 GFP sorts showed this problem. After todays sort, which had terrible fanning, I put on a sample of unstained fixed PBL's to see if it was instrument related. Not surprisingly, that sample gave great side streams. Obviously the problem is with the sample. I've never (never say never!) seen a fanning problem with surface stained cells and I don't understand what could be causing this to happen with GFP cells? Could the transfection method somehow "charge" the cells which interferes with the charge applied for sorting? As far as I can tell the media (which is MEM with Pen, strep, L-Glut, FCS, bicarb.) shouldn't do anything? Any help or suggestions would be greatly appreciated! Thanks in advance. Andy ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Andy Oberyszyn, M.S. Core lab manager The Ohio State University University Cell Analysis & Sorting Core 424 Davis Heart & Lung Institute 473 West 12th Avenue Columbus, Ohio 43210 Tel: 614/292-FLOW(3569) Fax: 614/292-7335 E-Mail: cytometry@osu.edu Website: http://heartlung.osu.edu/hlri/corelabs/flowcore.jsp ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ "What if the Hokey Pokey is really what it's all about?!?"
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