>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. Your observations sound consistent with what I have heard from other sites, and from my own experience. We also run PE-TR as our third color off of the 488nm argon line and have quite good results with it. We improved our measurements by changing the standard BD splitter you listed for a 600SP beamsplitter. The 610SP reflects approximately 50% of the light at 610nm; it isn't until you are at 615nm that you are reflecting near 90%. This cuts out a big chunk of the TR emission. Lowering the splitter to 600nm helps the TR and hardly affects PE. One problem with PE-TR is the limited availability of commercial conjugates. We use the Beckman-Coulter ECD, but find it costs about twice as much as other conjugations. Also, not many ligands are conjugated to it. > >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? I think you need to do some education here. PE-Cy5 is a great fluorochrome for a three-color Calibur. I think if you stained some cells with a biotinilated antibody and then stained half with a PE-Cy5 streptavidin and half with an ECD streptavidin and ran them on the Calibur, the difference would be blazingly obvious. Marty > >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 -- Marty Bigos Director, Flow Core Gladstone Institute of Virology and Immunology Building 3 SFGH Rm 509 415-695-3832
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