Len, >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? Yes, you do get weak emission from APC in the FL3 from 488. Not only that, there's no way to compensate it out except using software (FlowJo on Mac or WinList). The problem can be significant if you use a very bright reagent, like CD8 or CD45 isoforms. If you don't want to have to turn to software compensation, you can achieve less spillover by using some other reagent combinations (that are unfortunately inferior in terms of brightness). Instead of APC, you could use Cy7APC (sold by a variety of companies, but in limited antibody flavors), or Cy5.5APC (you have to make yourself). Or, instead of Cy5PE, you could use Cy5.5PerCP (sold in limited flavors by BD/PharMingen), Cy7PE (sold by various companies), or Cy5.5PE (you have to make it yourself). By switching the filters to accomodate either or these new combinations, you end up with much less spillover. I would recommend that you get filter sets to accomodate multiple combinations (i.e., in addition to your current filters, also get one for Cy5.5 (Cy5.5PerCP) and one for Cy7 (can use for either Cy7PE or Cy7APC)). Then, as you develop more applications using these newer reagents you can switch in and out as needed--or if you need the superior brightness of Cy5PE and APC over the other ones, and are willing to do the software compensation, stay with the standard set. mr
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:22 EST