Wayne, The problem lies in the cells size to nozzle size ratio. It should be at a minimum about 1:3 ratio with a 1:4 or 5 even better. When the embryo reaches the bottom of the sort stream, it causes the droplet to breakoff sooner then it should and therefore the saline droplet that has the cell in it is only partially charged. If this happens then the deflection of the partially charged droplet is suboptimal and the droplet never deflects far enough from the center stream to reach the collection tube. There is an old paper (1977) about particle size effect on the droplet breakoff written by Richard Stovel. Look up Journal of Histochemistry and Cytochemistry Vol. 25 No.7 pp813-820 Good luck. Glenn Glenn Paradis MIT Flow Cytometry Core Facility E18-463 50 Ames Street Cambridge, MA 02139 Voice: (617) 253-6454 Fax: (617) 253-3714 Email: gap@mit.edu > An investigator here wants to sort C. elegans embryos expressing >GFP. The embryos are anywhere between the 28 cell to 600 cell stage with >most closer to the smaller size. Each embryo is roughly 50x30 microns, >ellipsoid in shape with a fairly rigid shell encasing the embryo. We are >told that the embryos are relatively resistant to physical trauma. > We have run these on a Vantage with a 100 micron tip (no Macro Sort) >and can detect the GFP expressing embryos. Our problem arises with the >sorting; if we sort 40,000 GFP positive embryos, one only finds about 100 in >the collection tube. The good news is that those 100 are healthy in that >they grow normally following the sorting process. The question is, >what might cause this abysmal sorting efficiency? When we sort "normal" >cells like murine stem cells, lymphocytes, etc. we get very good recovery >and purity. If there is anyone out there with experience sorting something >like this I would greatly appreciate your suggestions. > >Thanks > >Wayne F. Green, Ph.D. >HCI Flow Cytometry Core Lab >wayne.green@hci.utah.edu
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