Jackie - If your investigator is using green excitation to view his RFP cells in the microscope, it is not surprising that he can see them there and you obtain a very weak signal using 488 nm excitation on a flow instrument; the microscope is probably better matched to the dye than the cytometer. You might try to optimize your detection filter for the RFP measurement; a 560 nm long pass in that detection channel would work best. From looking at the table below, RFP is one of the poorer GFP variants. For two color reporter gene assays we have successfully used GFP-YFP and CFP-YFP. The first pair can be used with a single argon 488 nm laser line. However, there is spectral overlap between the two emissions, so good controls are required, as well as a replacement for the normal FITC/PE filter set for detection. The CFP-YFP pair have good spectral separation, but require a violet-enhanced krypton laser for successful use. Other experimenters have had success using BFP excited by the UV lines of an argon laser. However, cellular autofluorescence increases with this excitation, and BFP is not as good as GFP or YFP. Marty Bigos Gladstone Flow Core Characteristics of Various Caltag GFP Variants Ex. Max Em max Extinction Coeff Quantum Yield DsRed 553 583 22,500 0.29 EYFP 514 527 84,000 0.61 EGFP 489 508 55,000 0.6 ECFP 434 477 26,000 0.4 EBFP 380 440 31,000 0.13 >Dear Flowers: > >I have an investigator here who is doing cotransfection using YFP/RFP. Under >the >microscope he can clearly see the positive RFP cells. However maximal >excitation >for RFP is about 585 so I 'm getting a very weak signal in the FL2 channel. >Also >the YFP signal is so bright it tails into the double positive quadrant. What >is the >best combination of fluorescent proteins to use to get optimal separation? >Thanks in advance for your help. > >Jackie Saleh >Aventis Pharmaceuticals >jacqueline.saleh@aventis.com
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