Maciej, The answer to your first question can be found at the Molecular Probes website <www.probes.com>. For rhodamine 123 (in methanol), the excitation max is at 507 nm, and the emmission max is at 529 nm. Comparing the spectra of rhodamine 123 and fluorescein, you can see that rhodamine 123 fluorescence is "red-shifted" by 10-15 nm; rhodamine 123 shows up in FL1 but you will see more "spillover" in FL2 than you would see with fluorescein. (Both appear to have about the same fluorescence at 650 nm, so the spillover to FL3 should be similar.) Although rhodamine 123 is better excited with the 514 nm argon line (79% of max) than the 488 nm argon line (44% of max), in our hands we saw better separation between unstained cells and rhodamine 123-stained cells on a FACScan (with 488 nm excitation) than on a FACStar (with 514 nm excitation). The superior fluorescence collection optics of the 'Scan made up for the inferior excitation wavelength. Good luck, Eric >Hello everyone. First, a newbie dye-chemistry question: >is the emission for Rhodamine 123 similar to PE or FITC or PerCP? >Assuming the basic FACScalibur laser configuration ( I don't remember >the wavelenghts). > >I tried to resolve monocytes using a perCP cd4 antibody. In >"unstimulated" (as in background level of activation) PBMC's CD4 >can resolve between lymphocytes and monocytes b/c of level of >expression. I know there are more efficient ways of doing this >4ex. CD14 and CD33. But in this experiment (oxidative burst assay) >I was not able to detect the FL3 signal at all! It was suggested >thereafter that CD4 gets dropped during activation and that could >be my problem. > >Has anyone tried adding a PerCP marker to the burst test to enhance >gating? I would apprieciate any suggestions. > >Maciej > >===== >`---------------------------------------------` >| Maciej S. Simm | 525 E 68th Street | >| Research Technician | Room N-805 | >| Cornell Medical Center | Tel. 212.746.3428 | >`---------------------------------------------` >__________________________________________________ >Do You Yahoo!? >Bid and sell for free at http://auctions.yahoo.com /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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