Try moving the fluorescence height adjust control a little bit to see if FL3 improves. It could just be an alignment problem. On occasion, I've had to sacrifice a little FL1 signal to boost FL3. If you have to move it a lot, tell your engineer to do an alignment to fix things. In the future pay attention to FL3 while you are doing daily alignment. This has saved me some grief. David McFarland SmithKline Beecham Pharmaceuticals ---------------------- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 12/20/2000 18:09 --------------------------- ANDREW.MORSCHAUSER@spcorp.com on 19-Dec-2000 14:04 To: cyto-inbox cc: (bcc: David C McFarland/DEV/PHRD/SB_PLC) 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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