Often a good solution to the problem of having only a fluorescein direct conjugate available is to use R-phycoerythrin anti-fluorescein to simultaneously quench almost all of the green fluorescein fluorescence, switch the emission to that of R-PE and significantly amplify the signal. Note that the fluorescein fluorescence below was collected in the R-PE channel and would have been perhaps 10-fold higher if collected in the fluorescein channel. We have not made tandem conjugates of phycobiliproteins with anti-fluorescein but this could presumably shift the emission further out yet. Figure 7.63 Color-shifting using a labeled anti-fluorescein/Oregon Green antibody. Jurkat cells were first stained with a primary mouse anti–human CD3 antibody, followed by fluorescein goat anti–mouse IgG antibody (F-2761), with the resultant fluorescence detected in the R-phycoerythrin (red-orange fluorescence) channel of a flow cytometer (blue curve). The weak signal was then shifted to better suit the R-phycoerythrin channel by the addition of an R-phycoerythrin conjugate of anti–fluorescein/Oregon Green antibody (A-21250). The resulting signal intensity is approximately two orders of magnitude greater (red curve) than the direct fluorescence from the first staining step (blue curve). [Image] Gerhard Nebe-von-Caron wrote: > I actually use Chlamydia as a nice example to demonstrate the advantages for > multichannel triggering in the microbiology tutorial. I use the SYVA Microtrak > kit which it is unfortunately FITC labelled but sufficient to count elementary > bodies by flow cytometry. If you want to use a green nucleic acid stain you > might want to add a second step antibody of your choice. > > Gerhard Nebe-von-Caron > > Research Scientist > Applied Science & Technology Group > SEAC - Safety and Environmental Assurance Centre > Unilever Colworth, Sharnbrook, Bedfordshire, UK - MK44 1LQ > > Tel: +44 (0)1234 264822, Fax: +44 (0)1234 222552 > E- mailto:Gerhard.Nebe-von-Caron@unilever.com > > -----Original Message----- > From: Chance, Tim [SMTP:jtc3@CDC.GOV] > Sent: Tuesday, March 19, 2002 7:17 PM > To: Cytometry Mailing List > Subject: RE: Chlamydia antibodies > > Does anyone know of a source for flurochrome labeled MoAb to Chlamydia > bacteria. Preferable PI or PercP tagged. > Tim Chance > CDC
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