Dear Howard, I've been fiddling around with the 3 laser setup on the FACS Vantage SE a few times, recognizing the problem with the 488 bleeding through. The cause of this in our setting was the iris in front of the UV arm, which was mal positioned, needing to be adjusted upwards to align with the UV line mirror and blocking out the 488. When it comes to aligning beads, I've found that a mix of Align Flow 2.5um for 488, 6um for UV and 6um for 633 from Molecular Probes works really well. They give two populations in scatter, a negative and positive in 488, 633 and UV, so it is easy to use when optimizing s/n. I see no reason why this bead mix could not be used for control except that they probably will fall out of range in SSC after having it set up for cells, but you will at least get a control for fluorescence. Molecular Probes have a bead that is excited by 630660 nm and emit from 670nm to 720nm which might help setting up for APC-Cy7 since you need emission at least above 710nm. Hope this helps. /Carl-Magnus Carl-Magnus Högerkorp Dep. of Immuntechnology University of Lund Sweden > Från: "Robinson, Howard" <Howard.Robinson@joslin.harvard.edu> > Datum: Mon, 31 Jul 2000 16:12:50 -0400 > Till: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > Ämne: 3 questions > > > 1.)Attempting to use all the laser lines simultaneously has not been easy. > I try to resolve 8 peak rainbow beads (Spherotech)for the UV using a 450/65 > BP, the peaks are all scrunched together. I attempted to use a 488 blocker > with the 450/65 BP, because I thought the 488 was causing the problem, but > ended up loosing all my signal. I'm wondering if a 488 shortpass would work > in front of the UV pathway to keep out 488 from bleeding through. Does > anyone have any ideas? > > 2.)I'm also interested in finding a bead that I can run after setting the > PMT voltages and parameter gains with unstained cells. I'd like to have a > fluorescent particle control that I run at the beginning and end of each set > of samples, utilizing all the settings I run the samples under, to ensure my > customers the machine is not causing any anomalies that may arise from time > to time in the experiments. I would like to have a negative bead and a > positive bead that is excited by a UV line, 488 line, and 633 line. Can > anyone offer me any advice? > > 3.) Lastly, I'm looking for an alignment bead for when I set-up to run > APC-Cy7. Does anyone know of a bead that emits at 767 nm and excited by a > 633nm laser line? > > > > Thanks, > > Howard > > > Howard Robinson > Flow Cytometry Core Facility > Joslin Diabetes Center > Room 491 > One Joslin Place > Boston, MA 02215 > > Howard.Robinson@Joslin.Harvard.Edu > (617)-732-2400 (Ext. 4516) > > >
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