The LSR has been in the lab for about 15 months now (it was brought in as a beta test). A wide range of applications have been run in 1, 2, and/or 3 laser mode. A presentation at ISAC XX summarized the applications through the spring of this year. I will email the PowerPoint file to whomever requests it. There are two papers in preparation. One with Jim Houston at St. Jude's on a general overview of the instrument, and the other describing a clinical application looking at evaluation of cytokine levels in live cells combined with surface markers for activation antigen expression level as well as cell phenotyping; look for it in J. Immunol. Methods early next year. Once set up properly (requires some thought), you can save CellQuest applications to disk that can then be retrieved and used by the most naive of users. Things that used to require a sorter (dumb idea if you're not sorting) and technician time can now be done self service and any hour of the day or night. Some of illustrations of applications are on the lab web page at: http://nucleus.immunol.washington.edu/Research_facilities/Lsr1/lsr.htm It's really out of date, but it will give an idea of what can be done. DNA with 4 surface markers (DAPI: pulse area, pulse width, FITC, PE, PE/TexRed, PE/Cy5) have been routine as have calcium flux with indo-1 and multiple surface markers. 5 colors for surface marker analysis and live/dead discrimination are routine. Useful combinations include: FITC, PE, PE/TxRed, and PerCP or PE/Cy5; TO-PRO-3 works well for live/dead discrimination. Alternatively, FITC, PE, PerCP or PE/Cy5, and APC; PI for live/dead. Note that PerCP is preferred since there is no cross-laser compensation issue as with APC and PE/Cy5. (Note too, that directly labeled or StrAv-Cy5 labeling is very good as a substitute for APC.) The early conjugates using PE/Cy7 have been almost useful, but that was some time back. Newer ones may be better. APC/Cy7 should work as well, but due lack of proper filters, it's on the list of things to test. With PMT signal splitting (very easy with BNC T-connectors) and some custom filters, you can squeeze 6 colors from the 488nm and 633nm lasers. Just today I finally got a dual band pass filter designed for Texas Red and PerCP/APC/Cy5 detection from one PMT. So far, uv-excited dyes for protein (AMCA for example, have been a bust). However, we're giving another try with Alexa 350. Although the dye is excited about half as well with 325nm light that the HeCd laser emits, it may work. The 325nm/441nm(?) Kimmon laser mentioned a while back is attractive. Where can I get one on loan? Where cross compensation is an issue, then the list mode data can be compensated in software after acquisition. There are three choices of programs to do this: WinList, FlowJo, and FCS Press. The latter is my current fave for its simplicity and ease of use, and the "appearance" of the compensated data. Also, for calcium flux data, recomputing the indo short/long emission allows you properly scale the ratio. FlowJo has the edge of terms of the kinetics computations and the ease of making graphical overlays of response curves. What else does the LSR need? The instrument absolutely SCREAMS/PLEADS/BEGS for the incorporation of digital electronics. (Hey Larry! It's time for another visit! I've got lots of DSP parts on the floor.) With that, all the PMTs could be used, pulse processing simplified, and more parameters could be collected for each sample. Right now, 9 parameter data files of multi MB size are common. Start thinking about a DVD-RAM drive for archives. The removable 5.2GB "personal" data disk is available today. (But who can forget those piles of 8" floppy disks?) Dave ====================== David M. Coder, Ph.D. Director, Cell Analysis Facility Univ. of Washington School of Medicine 1959 NE Pacific Street H474A Box 357650 Seattle WA 98195 tel. 206-685-3014 email: dcoder@u.washington.edu ----- Original Message ----- From: Calman Prussin <CPRUSSIN@niaid.nih.gov> To: cyto-inbox Sent: Wednesday, December 20, 2000 8:03 AM Subject: Additional flurochromes on the LSR Has anyone had experience with any antibody or avidin bound fluorochromes on the BD 3 laser LSR cytometer? The UV laser is a HeCad with a line at 325. Any thoughts or experience are welcome. Calman
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