Dear Sasha, It sounds like you have a Vantage SE optical setup, but might be over-driving the MacroSort head if you have so much noise from the hi-speed conditions. At 66KHz, for example, you ought to be able to get good scatter (FSC and SSC) with the drops breaking off near 32 to 35 drops from laser 1 intersect. Is your system a Vantage SE, or a Vantage with TSO and SE optics? There is a significant difference. We do PharRed/APC off 647nm krypton in the 2nd laser position on both our Vantage SE TSO systems using a 710LP to reflect the APC signal into a 670/14 and pass the PharRed (Cy7-APC) to a 787/42 (or 780/60) BP filter. The 3 way splitter remains and the 710LP sits between FL4 and FL5. There are some things you can do to minimize the signal losses at higher speeds (less time in the laser beam, etc.), feel free to email directly if you want to compare notes. Best Regards, Joe On Tue, 27 Apr 1999, Dr S Sreckovic wrote: > > Dear Ann, > > Re 1. > I must admit that I have a somewhat unique optical configuration > thanks to the guys from BD. I have 3 laser lines conf. with 3-way > beam-splitter. FL-1 and FL-2 are at the end of the main line, then only > FL-3 to the right (nothing at the opposite) and then 2 PMTs from UV to > the left and 2 PMTs to the opposite that I can use from the 633 laser > (between them is a 3-way beam splitter). I thought, if I use DM 710 SP > in the place of the 3-way BS and transmit <710 nm to FL1,2 and 3 and > reflect >710 to the left to FL-4, or I thought to assign it to FL-6 due > to possible compensation with FL-3. > > Re 2. > First, I think it is important to detect RBCs if you don't want them > in collection tubes, because if the instrument does not detect them > they can end up in the waste as well as in the collection tubes (am I > right?) > Secondly, I must go to the log FSC to register all RBCs and then > (especially on high pressures) I have to play with FSC and even SSC > obscuration bars to eliminate, as you said, monstrous noise from the > interfering (then 65 kHZ) drop drive. > It could be that because of wider obscuration bars for high-speed > sorting, FSC has been diminished on my instrument. > Unfortunately, researchers have broken collection tubes in a fuge so I > don't have any information on cell numbers, but next time I am going > to take a sample of sorted cells before I give it to them. > > Kind regards, > Sasha. > > > On Tue, 27 Apr 1999 14:18:44 +0000 Ann Atzberger > <Ann.Atzberger@EMBL-Heidelberg.de> wrote: > > > Hello Sasa, > > > > I also have a Vantage with an optical configuration for 2 lasers. The Fl-3 > > and Fl-4 PMT's are at opposite ends to each other. So with the help of a DM > > I can only reflect the emitted light of one laser to only one of these > > PMT,s. Am I right flow-ers?? > > Are you using two 488 lines? Is the optical configuration on your machine > > different? > > > > On your 2nd question: > > > > using linear settings on FSC/SSC should give you a very nice (typical) > > profile distinguishing granulocytes, lymphs, rbc,s. Threshold about 60-100. > > When I put FSC on log with ND out, I get a monstrous noise signal. I avoid > > working with FSC log as half the time I can't distinguish between the noise > > and the signal from the cells. > > The abort rate depends also on the size of the population to be sorted. If > > there's a huge amount of RBC's you could try pushing up the threshold level > > above the signal > > of the RBC's. You do not need to be able to see the RBC's to sort the > > granulocytes. > > You dfid not mention purity or recovery. > > > > Is the fl loss on the turbosort only? which fluorochrome is it? > > > > > > Regards > > Ann > > EMBL > > Heidelberg > > ---------------------- > Dr Sasha Sreckovic > Dept Path & Micro > University of Bristol > University Walk > Bristol, BS8 1TD, UK > Sasa.Sreckovic@bristol.ac.uk > 0117-928-8606 > > > > Joe Trotter Director, Flow Cytometry Mailstop Imm-20 The Scripps Research Institute 10666 North Torrey Pines Rd. La Jolla, California 92037
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