At 12:07 AM 11/9/01 -0500, you wrote: > >We recently acquired a BD LSR. Switching over from a Facscan >is a bit like patting your head and rubbing your stomach- in >the opposite directions. The visual cues are a bit off from >the usual Fascan display, and I'm having trouble getting my bearings. > >Running your standard lysed whole blood prep, I find it >practically impossible to achieve anything close to the >"Facscan traditional" 3-part Dif display. At the most I can >get Lymphs & monos, but no grans. If I manage to have all >three in the view- then the monos overlap the lymphs and >cannot be gated separately. The position of the FSC obscurator bar on this instrument is critical for good FSC. We always run ours with that plastic box covering the PMT's removed and thus can easily tweak the FSC bar. Give it a try, it's easy and may improve things a lot. >More disturbing (to me) is that I cannot get the lymphs off >the SSC low axis. I've pumped up the PMT voltage, then tried >toggling throught the 2-4-8 log scale. All I've managed to >so was increase the spread between the populations, or smear >out the lymphs- but the population itself will NOT move up >the axis. What am I doing wrong? > The SSC is a different matter, as SSC in this instrument is collected via "the Hubble optic" (and all that is implied by that name...) which basically picks up any light that it bouncing around in the filter block. Thus things like the position of the steering dichroics (510lp & 555lp) affect the amount of SSC signal as does the characteristics of the filter in the FL4 PMT position (try removing it and watch your SSC). When all these filters are positionally "optimised" for your application then still don't be surprised to need to use over 700V and an amp gain of 2 or 4 to get reasonable side scatter. >I'm also having a heck of a time comping the APC from the >FL3 channel (using FlowJo). All the other parameters look >fine, this one won't budge. If anyone has any special >tips-please pass them on! > Lastly, we have had a lot of success with retrospective compensation with the latest version of Winlist as you can adjust the compensation matrix while viewing the medians/means of the respective populations. Hope this helps, Geoffrey Osborne Specialist, Flow Cytometry, John Curtin School of Medical Research, The Australian National University, Canberra, 0200, ACT. AUSTRALIA email: geoff.osborne@anu.edu.au http://jcsmr.anu.edu.au/facslab/facshome.html (61 2) 6125 3694.
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:39 EST