I would like to thank the dozens of people that responded to my posting privately as well as on this forum. The problem with the FL3 was an extra 610SP DM was installed in the optical path and was replaced with the correct half mirror. With the new optical trunk for SE upgrade, the additional 610SP was sending the FL3 signal down a path where there is no longer a detector. Thanks again, Andy Morschauser Schering-Plough Research Institute Department of Immunology 2015 Galloping Hill Road K15-3-3945 Kenilworth, NJ 07033 908-740-3089 ____________________________________________________________________________ ___ Subject: Decreased signal in FL3 with SE upgrade From: ANDREW MORSCHAUSER Date: 12/19/00 2:04 PM 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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