Hi Flowers - The filter change that Lora suggests should work well provided you are not using a dye laser on the instrument. Then the PE channel would be suseptable to picking up scattered light from that laser (595 nm to 605 nm is the typical range used by the dye lasers in flow applications). With a HeNe laser there should be no problem. The use of the 575/25 filters stems from the historical development of PE (and APC) as new fluorochromes. At the time the "advanced" systems were two color ones using FITC excited at 488nm and Texas Red excited by a dye laser at 595 nm. When PE conjugates were first made, they were added on as a third color. The 575/25 was chosen to block scatter from the dye laser and minimize spectral overlap with FITC. Similar considerations led to the use of a narrow bandpass for APC. For those instruments using a HeNe laser for 4th (APC) and 5th (Cy7APC) colors, it is clear, as Lora pointed out, that there are better filter choices for both PE and APC than the historical ones. I also want to mention that the use of a "better" filter in the analyzers is not the only reason why PE measurements on these instruments are so much better than jet-in-air sorters. First the analyzers have much better light collection optics with the immersion flow cell. Second, the flow rate is slower, allowing a much longer integration time for the measurement. And lastly, the low power on the analyzer allows for multiple excitations of each PE fluorochrome, while the much higher power used on jet-in-air instruments puts the PE into some unavailable state which lasts a (relatively) long time. Thus one gets a much better signal to background measurement with PE using the low power analyzer. >Dear FACS Vantage Operators/Users, > >We have discovered recently that we had been running our Vantage with a >different filter set on FL2 than what is issued on both the FACScan and the >FACSCalibur. We had been running with a 575BP26 for quite a while to >analyze PE. All of our work "seemed" OK; bright markers were only slightly >dimmer than what was seen on the analysis machines. But when we needed to >do some dim marker sorts and were coming up with negative expression, we had >a problem. So, we ran some tests by carefully placing the isotype control in >the same channels on all machines. The Vantage (with the 575BP26 installed) >gave us results that were dimmer than the Calibur by 192 channels! Then when >I installed a 585BP42, which is the same filter used in the Calibur, I was >only 62 channels dimmer. The 62 channel difference was also seen on FL1, so >I felt the drop was due to laser alignment and stream in air issues. What a >relief!! > >I kick myself for not checking the filter sets earlier. I discussed these >issues with my field engineers and we agreed that a 575BP26 should work for >PE, but really, it doesn't. I was loosing a lot of sensitivity!!! > >Hoping others may learn from my experiences, I'm giving the following >advice: be sure to run PE with 585BP42 filter set in FL2 on the >FACSVantage, when using an Enterprise Argon/UV laser. > >Thanks for your time, > >Lora Barsky >Children's Hospital Los Angeles >Research Immunology/BMT >323 669-5935 Marty Bigos Stanford Shared FACS Facility bigos@stanford.edu
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