> Hi Andy, > what are the gfp tagged cells? if they are a substrate attached cell line, > they are substantially larger than pblšs to sort. You need to make bigger > drops. Try bumping up the nozzle size (that will involve slower speeds and > only do it if you can get decent side stream separation) or as a starter try > to sort on normal R mode with 1.0 drops and see if that cleans up the side > streams with the cells running through. With the cells running, also gently > tweek the amplitude and that might also help clean it up. > hope this helps and good luck > > > 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?!?" > -- Gisele Knowles >>Director Centre for Cytometry and Scanning Microscopy Sunnybrook and Women's Research Institute Room B 210 2075 Bayview Avenue Toronto, ON 416.480.6100x7282 (office) x7284/5 (lab)
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