I didn't see anyone else respond to Franck Morel's query about the B-D LSR, so I'll put my two cents in. Having been tied to a sorter for UV applications for the last decade (a shameful misuse of both sorter and sorter-operator [i.e., my] time), all I can say is that it's about time. We have had our instrument for about 3-4 months, and at times (see below) the data have been incredibly good, with DNA cv's of 3-5% and no discernable noise (HeCd lasers must have improved significantly). We routinely perform 3-4 color surface/cytoplasmic immunfluorescence together with DNA (UV) analysis. On the sorter, we used DAPI, FITC, PE, Texas Red, and APC. On the LSR, we presently use DAPI, FITC, PE, Red-613, and PerCP (we have the UV/488 model). When the red laser is available/installed, we will be able to use DAPI/FITC/PE/PerCP/APC (a combination we presently use, sans DAPI, on the Calibur), and most remarkably about this combination, the only compensations are FITC/PE. Needless to say, given this and the absence of daily alignment, everyone in my lab is able to do experiments without the need for me to be there, which represents a major increase in productivity, not to mention freeing up the sorter for what it is intended to do. Calcium flux experiments have also been almost trivial to set up; although the people doing this (from other labs) have not actually SEEN flux, I think this is a biological problem, because the baseline looks quite normal. On the cautionary side, on three occasions we have experienced significant deterioration of laser alignment (to give credit where due, B-D has always responded by sending an engineer within 24-48 hours). The source of the problem has been difficult to pinpoint, and may be due to the instrument getting settled in, or may have a more deep-rooted source, only time will tell. Right now I can say that it's been a major improvement for us, and assuming that the stability problems are worked out, I think it's going to be an extremely powerful resource, as well as freeing up sorters for sorting. In my opinion, the usefulness of this instrument, either in 2 laser (UV/488) or 3 laser form is dramatically enhanced by the ability to use PerCP, given the simplicity of compensation mentioned above. Of course, this is limited by B-D/PharMingen's willingness to conjugate antibodies to PerCP (hint)... It is true that this instrument is NOT inexpensive, but I think that when you balance the lack of need for an operator and the time that is made available on the sorter, it becomes justifiable. Howard T. Petrie, Ph.D. Head, Developmental Immunology Laboratory Director, Monoclonal Antibody Core Facility Memorial Sloan-Kettering Cancer Center Box 341, 1275 York Avenue New York, NY 10021 phone (212)639-2149 fax (212)794-4019
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