Hi Lora... Maybe your users originally used ECD / PE-Texas Red on an older FACScan or FACSCalibur - our rather "ancient" FACScan (1987) has a 650 LP in front of the FL3 detector, and PE-Texas Red conjugates work acceptably on it for dense antigens, losing less than a log decade signal on average compared to PE-Cy5. I think our FACSCalibur (also somewhat older, 1994) also has a 650 LP on the FL3 detector. If the newer FACSCaliburs now have a 675 LP in front of FL3, PE-Texas Red would be far less useful for this particular instrument. Anyway, you are absolutely right - why use PE-Texas Red on a Calibur and risk losing signal intensity when so many PE-Cy5 conjugates are available. Your users should save ECD for the Vantage or for Beckman-Coulter instruments, which are designed for it Take care, Bill At 05:51 PM 11/14/2001 -0800, Barsky, Lora wrote: >Hi everyone, > >In our quest to run more colors on the sorter, we have been using Coulter's >ECD (PE-TxRed) conjugates on our Vantage SE. The vantage is equipped with >Enterprise and HeNe lasers. We do not have any means to cross-beam >compensate. So, plugging the Bandpass for RED613 (610bp20) into FL3 has >made it possible to sort four colors on our system along with the >traditional, FITC, PE and APC. We have also chosen ECD because PerCP >quenches about 1.5logs on our Vantage and PE-Cy5 crosses too much into APC. > >BUT... I have users here who want it to work on the Calibur, because they >don't want to buy or can't buy PerCP conjugates or PE-Cy5 conjugates and >because they want to directly compare emissions from both machines. But that >is another problem of mine - I digress... So, since we cannot change out the >optics for FL3 on the Calibur, they are collecting only the far red tail of >the emission with the standard 675LPfilter; they are completely missing the >emission peak. I've told them that the conjugate wasn't designed to work on >a Calibur, but the user "saw signals" and now I'm dealing with their "it >works fine for me" syndrome, when I ask them to refrain from promoting this >antibody combination to other users who are beginning 4 color work. Could >anyone provide me references that would help get my point across? > >If there is someone out there who does this combination successfully on >their Calibur and really trusts their data, could you share with me the >typical instrument settings values you're coming up with? I understand that >there are instrument to instrument differences but would still like to >know... > >Thanks, >Lora W. Barsky, Research Specialist >CHILDRENS HOSPITAL LOS ANGELES >Research Immunology/Bone Marrow Transplant >Flow Cytometry Core >4650 Sunset Blvd. Mail Stop #62 >Los Angeles, CA 90027 >(323) 669-5935
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:40 EST