Dear Andrew, About a year ago I did the same type of comparisons. However, I didn't see those differences between the Vantage and the Vantage SE. To optimize sensitivity on the SE in general I did however also play around a bit with the PMTs by twisting them in their holder and also by turning the holder itself. This gave better optimized S/N on all parameters. Also worth trying is to evaluate the performance of each PMT in the FL1 position, and them position them according to where you need the best sensitivity. Even if these tricks may improve sensitivity, your problem sounds more like electronics related. I would probably be persistent in getting the BD technicians there to fix it. Carl-Magnus Högerkorp Dept. of Immunotechnology University of Lund Sweden -----Original Message----- From: MORSCHAUSER, ANDREW [mailto:ANDREW.MORSCHAUSER@spcorp.com] Sent: Tuesday, December 19, 2000 8:04 PM To: cyto-inbox Subject: Decreased signal in FL3 with SE upgrade This message is directed towards FACSVantage users that have upgraded their machine from a FACSVantage to a FACSVantageSE w/Turbosort. I am experiencing a decreased sensitivity in FL-3. When comparing alignment particles (cRBC, Alignflow beads, Calibrite beads) between a FACSVantage, FACSVantageSE w/TSO and a FACSCalibur, results have shown the FACSVantage SE signal to be significantly lower than the Vantage or Calibur. Furthermore, when looking at splenocytes stained with CD4 PE-Cy7 (787/40 filter), there is no discrimination between negative/unstained cells vs. stained cells. The signals on the FACSVantage and Calibur are nearing the third decade while the FACSVantageSE signal was negative with log amps and voltages greater than 900V. I tried increasing the log offset as far as it will go and found little if any improvement in the signal. Boards have been replaced and new PMTs in FL3 have done nothing to remedy the problem. BD has been very responsive to my concerns, but the problem is still present and I was wondering if anyone else has experienced similar problems with the extra mirror and different optical path for a 5-color FACSVantage SE. Any comments or suggestions would be greatly appreciated. Andy Morschauser Schering-Plough Research Institute Department of Immunology 2015 Galloping Hill Road K15-3-3945 Kenilworth, NJ 07033 908-740-3089
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