Hello Henry... Teresa and Bob Hawley (ARC Holland Lab) and I wrote the following piece for the list a few months ago. Bill ______________________________ Teresa and Bob Hawley at the American Red Cross Holland Lab have been using DsRed extensively and designing filter configurations for it (with and without other FPs) on our Vantage SE at the NIH. All of our collective comments are below. First the instrument end (with edits from Teresa)... First, we can excite DsRed reasonably well with a 488 nm line and detect it through a 585/42 or 610/20 filter. GFP and DsRed can therefore be run simultaneously on a FACScan or FACSCalibur with the standard FL1/FL2 filter setup. We can also run DsRed simultaneously with EGFP and EYFP on a Vantage using an I-90 laser emitting at 488 nm, the standard Omega GFP/YFP filter/dichroic set (525 LP splitter, 510/20 for GFP in FL1, 550/30 for YFP in FL2) and a 560 SP and 585/42 or 610/20 filter to split off and detect the DsRed signal respectively (in the FL3 PMT). Ar laser lines at 514.5 nm and Kr at 520 and 530 nm give improved DsRed excitation. However, the optimal excitation source for DsRed in our hands is a yellow laser line - we use our I302 krypton laser tuned to 568 nm (35 mW), and detect DsRed through a 610/30 filter with a sharp cutoff at 595 nm (Chroma) to reduce scattered laser light in the PMT. The standard 610/20 filter BD supplies will also work with slightly higher noise. The DsRed signal is detected on the third signal path PMT (FL4). Teresa has also been doing 4-color analysis with GFP, YFP, CFP and DsRed using the argon 458 line as the primary laser as described by Beavis and Kalejta (Cytometry 37:68-73, 1999) and the Kr 568 nm line as the tertiary laser. GFP and YFP are collected as above - we use a 500 LP to split off the CFP signal, collected though a 485/22 filter (FL3). We have been able to analyze GFP/YFP/CFP and DsRed this way simultaneously without 568 nm laser light leaking into the YFP PMT (FL2), since the primary and tertiary beams and signals are sufficiently separated -however, the instrument alignment has to be perfect and is hard to reproduce from day to day. We get more consistent results with a 568 nm blocking filter in front of the YFP PMT (FL2). Considerable intra- and interbeam compensation (and teeth-gritting instrument alignments) are required to run multiple FPs, but it can be done. Second, here are Teresa and Bob's comments on applications... "DsRed's relatively low extinction coefficient and quantum yield appear to make it inappropriate for the study of expression at the level of single gene copy in most instances. However, we feel that in situations where high expression can be achieved (due to a strong promoter or integration of multiple gene copies), DsRed's red-shifted spectrum makes it rather attractive. At present, the best application is in vivo tracking of stably marked cells (probably isolated by sorting) which can be utilized in conjunction with similarly engineered cells expressing the other FPs or surface-labeling antigens." So, DsRed in its present incarnation is certainly possible to use on a FACScan/FACSCalibur or Vantage, but its extinction coefficient and yield are lower the GFP. Nevertheless, its EX/EM characteristics are potentially very useful for multiple FP experiments or immunophenotyping experiments. I'm sure the good folks at ClonTech are engineering improvements as we speak. Bill Telford NCI Medicine Branch Flow Cytometry Core Laboratory DCS-NCI-NIH ____________________________________ At 05:41 PM 10/23/00 -0400, you wrote: > >Help with an old question, please. > >Are there people out there who have had success detecting a minor >propulsion of dsRed + mammalian cells with a larger population of eGFP+ >cells? Is anyone doing this with a 488 laser? Is this only possible >with specific filter sets? > >Henry H. Wortis, M.D., Professor >Department of Pathology, >Director, Graduate Program in Immunology >Tufts University School of Medicine >136 Harrison Ave. >Boston MA 02111 >Phone 617-636-6718 >FAX 617-636-2990
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