>Hello fellow flow-ers, > >I am operating FACSVantage SE at the University of Bristol and I have >some really practical questions that I hope someone is going to give >two pence for. > >1. Does anyone have experience in using FITC, PE, QuantumRed, and >PE-CY7 - all from 488 nm, in the same time on a Vantage. Is DM 710 SP >(before FL1, FL2 and FL-3 and reflecting to FL-4) sufficient or should >I also need band-pass filter in front of FL-4 used for detection of >PE-Cy7? I have FL-6 installed from recently so if I use FL-6 for PE-Cy7 >detection I can even compensate between PE-Cy5 and PE-Cy7. > >2. I was sorting (believe it or not) only on FSC and SSC for one of my >researchers to sort pure (and untouched) granulocytes apart from few >lymphocytes and plenty of RBCs. It was a low-speed sort on 11 PSI, >with DDF - 26 kHZ and 2000-2500 events/sec. ND filter was out and FSC >set to log in order to detect RBCs and exclude them from sorting. >In this way I had more then 40% aborts and I assumed those are mainly >force-aborts so I increased dead time from original 7.5 and get lowest >abort rate on 13.5 but still more then 20%. Finally I had to put ND >filter in and lose some of RBCs from detection in order to get less >then 10 % abort rate. I thought that FSC amplification is going to >increase only peak of the pulse but not a time of its detection also, >having in mind force-aborts. I would appreciate any thoughts and I am >aware that the best thing is to lyse RBCs but I must comply with >researcher's aim. > >3. I am experiencing big fluorescence loss with TurboSort and more >then 10000 events/sec especially on FL-3. Anyone else? > >Thanks everyone. > >---------------------- >Dr Sasha Sreckovic >Dept Path & Micro >University of Bristol >University Walk >Bristol, BS8 1TD, UK >Sasa.Sreckovic@bristol.ac.uk >0117-928-8606 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
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