Richard, Unfortunately your choices cannot all work at once on this instrument as there is no way without special engineering modifications to use the detectors in any configuration other than a 4-0-2 or 3-1-2, meaning the UV has to be used to achieve 6 colors. To phrase it nicely, this is not a real optimal configuration. Randy T. Fischer NIH/NIAMS Building 10, Room 6D57 9000 Rockville Pike Bethesda, MD 20892 (301) 594-3537 fischer1@mail.nih.gov > ---------- > From: Richard Haugland > Sent: Wednesday, October 30, 2002 8:37 PM > To: Cytometry Mailing List > Subject: Re: Fluorochromes choice > > <<Message: Microsoft Outlook Message>> > I would recommend fluorescein (or Alexa Fluor 488), R-PE and an R-PE > Alexa Fluor 620 tandem conjugate off the 488 laser and APC with APC > Alexa Fluor 680 off the 633 nm laser. The Alexa Fluor 610 R-PE is > superior to Texas Red R-PE tandem conjugates because its emission is > moved about 20 nm toward longer wavelengths but it is barely excited at > 633 nm. One can presumably do five dyes off the 488 nm argon-ion laser. > You would not need the UV laser then. We have made Zenon Mouse antibody > labeling kits with all of these dyes, including the tandem conjugates. > > Figure 6.34 Normalized fluorescence emission spectra of 1) Alexa Fluor > 488 goat anti-mouse IgG antibody (A-11001), 2) R-phycoerythrin goat > anti-mouse IgG antibody (P-852), 3) Alexa Fluor 610-R-phycoerythrin goat > anti-mouse IgG antibody (A-20980), 4) Alexa Fluor 647-R-phycoerythrin > goat anti-mouse IgG antibody (A-20990) and 5) Alexa Fluor > 680-R-phycoerythrin goat anti-mouse IgG antibody (A-20983). The tandem > conjugates permit simultaneous multicolor labeling and detection of up > to five targets with excitation by a single excitation source - the 488 > nm spectral line of the argon-ion laser. > > > [Image] > > Figure 6.37 Normalized fluorescence emission spectra of 1) > allophycocyanin, crosslinked, goat anti-mouse IgG antibody (A-865), 2) > Alexa Fluor 680-allophycocyanin goat anti-mouse IgG antibody (A-21000), > 3) Alexa Fluor 700-allophycocyanin goat anti-mouse IgG antibody > (A-21003) and 4) Alexa Fluor 750 goat anti-mouse IgG antibody (A-21006). > The tandem conjugates permit simultaneous multicolor labeling and > detection of up to three targets with excitation by a single excitation > source - the 633 nm spectral line of the He-Ne laser. > > > [Image] > > > 380/LP > 400/40 > 424/44 > 500/11 > 510/20 possible filter for fluorescein or Alexa Fluor 488 > 530/28 > 575/15 filter for R-PE > 610/20 best of these filters for Alexa Fluor 610-R-PE but a 630/~15 > would be better > 660/13 > 670/LP probably best for Alexa Fluor 680 R-PE > 682/13 > > > > Michal Abel wrote: > > > > > Dear experts in flow cytometry, > > > > I have a possibility to use 6 colors FACS. The trouble is that I am > > not sure it is possible with the filters I have. > > I would very much appreciate any help. > > The instrument has three lasers: 325, 488 and 633 nm and I can use > > following filters: > > 380/LP > > 400/40 > > 424/44 > > 500/11 > > 510/20 possible filter for fluorescein or Alexa Fluor 488 > > 530/28 > > 575/15 filter for R-PE > > 610/20 best of these filters for Alexa Fluor 610-R-PE but a 630/20 > > would be better > > 660/13 > > 670/LP probably best for Alexa Fluor 680 R-PE > > 682/13 > > Is it possible to find 6 different flourochromes, which would be > > efficiently differentiated with these filters/lasers ? > > Thank you very much for your help. > > > > Michal > > > > -- > > > > Michal Abel, MD, PhDLaboratory of Clinical ImmunologyINSERM U25Necker > > Hospital, 161 Rue de Sèvres. Paristel: 33 1 42 19 28 87fax: 33 1 44 49 > > 53 74 > >
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