At 10:02 AM +1000 6/2/00, Len Brown wrote: >Dear All > >We are attempting to set up 4 colour assays on our FACS Vantage (NOT SE >upgraded) using APC on a HeNe laser delivering about 30 mW. Abs are from BD. > >1) Should we expect to see the APC signal in the FL3 channel (optimised for >PECY5) of our 488 nm line? ie. Is APC excited at all by the 488 nm laser? > >2) Is anybody using BD's CD11c-APC mAb on a similar system, if so how does >the signal compare with PE. > >Any help or hints on setting up 4 colour on a Vantage with 488 and 635 nm >laser lines would be most appreciated. > >Len Brown As has been discussed a bit before here, you will have a problem using PECY5 and APC. BD did not consider compensation between laser 1 and laser 2, even in the design of OmniComp. This is unlike the situation in the FACScalibur, where they did implement this and you can use those combinations. The problem arises because APC is in fact excited to a small but significant degree by the 488. The FL3 filters for PECY5 are perfect for APC emission, so you will see this signal in FL3 channel and will not be able to compensate it out. Since APC can be quite bright, the signal even at a few percent of maximum (i.e. the authentic signal in FL4) will be noticeable and could be troublesome. Workarounds: 1) Use APC and APC-Cy7. The latter is weak, but acceptable for bright stains. Make sure to have the right filters and a red-sensitive PMT. Since both are excited by laser 2 you can compensate. 2) PI can be used in FL3 since the weak APC cross-bleed will usually not be anything like the dead cells you will gate out. Just check to make sure in single colors. 3) CY5 in our hands does not seem to be nearly as well excited by the 488 and in some cases we have not seen the cross-bleed into FL3. However, this may just depend on the brightness of the reagent. You may want to check for yourself. We have tried the cryptofluors from Intergen and though they are bright and work well, they also bleed into FL3. I have heard that BD is working to fix this problem. Mark Shlomchik, MD, PhD Associate Professor of Laboratory Medicine and Immunobiology Yale University School of Medicine 203-688-2089 203-688-2748 (fax) mark.shlomchik@yale.edu
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