As he doesn't seem to be the only one with this problem, I'll relay to you all what I wrote to Andy: Andy- Until you mentioned that you had had the PMTs changed, I thought I had a suggestion. And it may still be something to check out. Apparently BD uses two different kinds of PMTs. One is more sensitive in the visible spectrum and is used for the regular fluorochromes, and the other is more sensitive in the UV range, and is used for that purpose. When we had our VantageSE installed, the service rep mixed them up and our FITC PMT was one of the UV kind. Needless to say, the sensitivity of that PMT was much less than for the PE etc. PMTs. When they switched them back the proper way, everything was fine. This may be something to look into. cheers and happy holidays- Regina Regina H. Harley, M.S. Lab Supervisor - Flow Cytometry Facility Center for Vaccine Development University of Maryland - Baltimore 685 West Baltimore Street Baltimore, MD 21201 (410) 706-7376 FAX: (410) 706-6205 rharley@medicine.umaryland.edu >>> Karim Vermaelen <Karim.Vermaelen@rug.ac.be> 12/21/00 03:40AM >>> Hi Andy, We've been experiencing similar problems since we had our Vantage upgraded to a Vantage SE : voltages for APC (P6/FL5) and APC-Cy7 (P5/FL4) detection have to be set abnormally high (>900) (we use optical bench configuration "6" as drawn in the manual). With our old "regular" settings (in the past we considered 600V amp to be quite high already), both stained as well as unstained cell clusters disappear down to oblivion. I'm sending some dot-plots of this situation to BD soon, at this time they don't know any answer to the problem. FL3 was down shortly after the SE upgrade. However after giving a little nudge to the FL1-FL2 vs. FL3 splitter (610 SP) the signal balance was restored in favour of FL3 without affecting FL1-FL2 detection. I assume you have checked your optical bench configuration in detail - it's easy to misplace a splitter, invert a filter etc... Refer strictly to the configurations drawn in the manual. If this doesn't help it's definitely up to BD to fix the problem. I will keep you posted if anything new pops up on that front. "MORSCHAUSER, ANDREW" wrote: > 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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