Having had one of the first LSR analyzers installed in the field, my experience parallels that noted below. For examples of data (4 color surface markers, DAPI and 3 color, DNA (DAPI) linearity checks, and calcium flux comparisons with the Vantage see the LSR lab web page: http://nucleus.immunol.washington.edu/Research_facilities/Lsr1/lsr.htm Note that the DAPI and 3 surface marker illustrations are from an 8-parameter data file. For a user-operated instrument, it is quite easy to set complex analytical procedures. Once defined, protocols, screen layouts, instrument configuration, and all instrument settings are easily retrieved from CellQuest disk files. There is no instrument alignment to be done. In fact, the optical stability has been boringly invariant. Allow about 30 minutes warm-up time if using the HeCd laser, and then retrieve your experimental setup and run samples. So, if anyone is familiar with a FACScan and CellQuest, then multi-color experiments on the LSR should be easy to run. Fluorochromes that have been used successfully include: FITC, PE, PE-Texas Red, PE-Cy5, PE-Cy7, PerCP, CSFE, GFP, DAPI, Hoechst 33342, PI, 7-AAD, and indo-1. Calcium flux experiments using indo-1 have been quite successful. As I note on the above web page, the resolution of populations showing different levels of calcium flux seems to be better on the LSR than the Vantage. In fact, the shorter calcium-bound fluorescence of indo-1 is better resolved on the LSR than the Vantage since calcium-bound indo-1 is excited more efficiently with the 325nm light of HeCd laser. On the other hand, the calcium-free indo-1 fluorescence is better resolved when excited with the longer wavelength uv lines of the argon laser. (I built a very cheap and simple water jacket to maintain samples at 37C. It consists of a 15mm diameter polypropylene test tube cut to a height to take a standard 13mm sample tube, and wound with two concentric layers of about ~3mm silicon tubing; electrical tape keeps the tubing windings in place. The ends of the tubing are connected to a recirculating 37C water bath. There is sufficient clearance for the sample arm to support the water jacket and sample tube. It's very easy to remove the sample, add agonist, and replace the sample tube. Note that the gap on the time vs. indo-1 ratio density plot is shorter on the LSR than on the Vantage.) As Dr. Petrie notes, it keeps the Vantage doing high-speed sorts, plate sorts, and 6 to 8 color analysis as it should be. If all goes well (still waiting for some filters to arrive), the HeNe laser I installed on the LSR may give us the ability to do DNA and 6 other colors in addition to pulse processing. Stay tuned . . . . Dave ********************************** David M. Coder, Ph.D. Director, Cell Analysis Facility Dept. of Immunology Univ. of Washington School Medicine Box 357650 Seattle WA 98195-7650 tel. 206-685-3014 fax. 206-543-3480 email: dcoder@u.washington.edu ----- Original Message ----- From: Dr. Howard Petrie <h-petrie@SKI.MSKCC.org> To: cyto-inbox Sent: Friday, March 03, 2000 12:32 PM Subject: re: BD LSR ? 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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