>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?!?" Hi Andy, You didn't say what size your GFP cells are and what nozzle size you are using. These two parameters are important. On my MoFlo I tend to stick to a rule of thumb that the cell diameter should be no more that 3/5ths. the size of the nozzle, max. If you go beyond that you will see poorly formed side streams. I'm sure that the same rules will apply to your Vantage. Your Peripheral blood cells are made up generally of small sized cells and it's no wonder that they do not interfere with the formation of your side streams. Hope this helps, Andy. -- Andy Riddell Flow and Imaging Lab CIMR Hills Road Cambridge UK page: 362 tel: (0)1223 762597 fax: (0)1223 336900 email: ar289@cam.ac.uk
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