Andy The fanning you describe sounds very much like it is a problem caused by small clumps of cells too small or loose to clog the nozzle orifice. As these clumps pass through the nozzle orifice they are disrupted into single cells (or smaller clumps), this disrupts the stable drop formation resulting in the production of many drops of differing sizes. The charge these a drop carries is dependent on its diameter and carrying a different charge they are accordingly deflected a different amount. In the case of a specimen containing a high percentage of these clumps it is possible to get what appears to be a continuous 'stable' fan, this fan will occur even when no sort gates are selected provided the deflection plates are turned on. You can check for this problem in two different ways. 1. Place a slide where you would normally place a tube, in bad cases you will get a straight line with out any obvious indication of a spot, under less extreme circumstances you will get the usual spot and a few satellite spots. 2. If you can observe the signal pulses on your cytometer, watch the forward scatter trace (any parameter will probably do). Besides the usual superimposed pulses for most signals at the left hand edge of the screen you will see a few pulses further to the right which occurred within the sweep time of the oscilloscope - this is fine, but when a small clump comes through and disrupts you will see a huge shower of pulses to the right, some will probably be of excessive magnitude. The solution is too filter or preferably disrupt these very small clumps. Sometimes you can actually see, with the unaided eye, these fine clumps in the sample tube as a sort of mist. Hope this helps. ############################################################################ Email: alan.bishop@imvs.sa.gov.au Tel: +61 8 82223602 Fax: +61 8 82323139 Mail: Alan Bishop MA cantab. Senior Flow Cytometrist PO Box 14 Rundle Mall Flow Cytometry Facility Hanson Institue IMVS ADELAIDE SA 5000 AUSTRALIA
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