Andy, You might want to try a bigger nozzle. Usually it's the size of the cells that produces fanning, once you rule out instrument setup, as you have. You need a nozzle diameter that's around 5 times the diameter of the largest particles in your sample. Most cultured cells are considerably larger than PBL's. Also, be sure clumps are kept to a minimum. (A little microscopy can go a long way here.) I often resuspend with a pipette immediately before installing a sample on the instrument rather than just trusting a vortexer. To rule out the media causing this (maybe there's some viscosity issue), try resuspending the cells in straight PBS, or at least "cutting" your sample by diluting it with PBS. Most cells will tolerate it for the duration of a sort. Good luck, Neal Andrew Oberyszyn wrote: > 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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