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
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