I'm sorry for the post that offended some people as "advertising." It was specifically intended to go directly to only the questioner but inadvertently went to "all". I usually try to post to the Board only those responses that I feel will be of general interest and are, in most cases, a solution or information that others are unlikely to give but make many other responses directly only to the individual. With regards the matter of 325-nm light-excitable dyes, Bill is right that there are few dyes useful for conjugation in this spectral region. Alexa Fluor 350 (AMCA has absorption and emission peaks about 8 nm longer than Alexa Fluor 350). [Spectra: 12KB] and Cascade Blue [Spectra: 12KB] These are normalized spectra and the extinction at 325 nm for each dye is perhaps 6000-8000 because Cascade Blue has a higher peak extinction. Pacific Blue is really quite long wavelength for the 325-nm laser and has emission stretching more into the >500 nm spectral range. [Spectra: 12KB] It is probable that 7-methoxycoumarin-3-carboxylic acid would make a good fluorophore (exc max ~336 nm, em max ~402 nm) with an extinction at 336 nm of about 20,000 but we have never seen an interest in this spectral range previously. 7-methoxycoumarin-4-acetic acid may be an even better match but I don't have those data. It is sometimes used as the fluorophore in quenched protease substrates. William Telford wrote: > Hello Michal... > > Although I agree with almost all the comments so far about doing > 6-color on the original LSR, you still might want to give the > UV-excited fluorochrome AMCA (or its more recent descendant Alexa > Fluor 350) a try as your sixth color. AMCA is actually a reasonably > bright fluorochrome - unfortunately its emission at ~450 nm falls in > an area of high cellular autofluorescence, making it appear dim > relative to background. However, if you use it to label a densely > expressed marker, it might be bright enough to visualize. We've made > it work in the past for a few highly expressed surface markers, such > as CD8 on mouse T cells - expect at least a one-to two log decrease in > fluorescence compared to FITC. Use your 424/44 filter for detection. > We have also tried AMCA and Alexa Fluor 350 with a variety of UV > lasers - although its emission peak is around 350 nm, the HeCad 325 nm > line still seems to excite it as well as (or no worse than, anyway) > the longer argon and krypton UV lines. You can use Alexa Fluor > 350-streptavidin to label a biotin-conjugated antibody, or use the > Zenon kit from MPI to make a "conjugate". > > From an instrument standpoint, you might want to get the 4-2-2 LSR > upgrade to do four colors off the 488 nm and two off the 633 nm. I > know this configuration exists for the LSR, although I'm not > absolutely sure if BD is routinely doing upgrades to it. Alternately, > you might want to consider investing in the 405 nm violet laser diode > upgrade and use Cascade or Pacific Blue as your sixth colors, which > give much better signal-to-noise ratios than AMCA. > > Enjoy, > > Bill Telford > NCI-NIH > > (and yes, no financial stake in Molecular Probes, BD Bioscience, or > anyone else for that matter!) > > At 11:21 AM 10/30/2002 -0800, 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 >> 530/28 >> 575/15 >> 610/20 >> 660/13 >> 670/LP >> 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, PhD >> Laboratory of Clinical Immunology >> INSERM U25 >> Necker Hospital, 161 Rue de Sèvres. Paris >> tel: 33 1 42 19 28 87 >> fax: 33 1 44 49 53 74 >
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