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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