Jeff, I think that the flow cytometry community in general has overemphasized the difficulties arising from the use of AO. Much (if not all) of this is unwarranted. In my opinion, AO is extremely useful for detecting perturbations to the cell cycle, perhaps second to BrdU. It's perhaps the best way to detect the transition from G0 to G1. Its advantage is that it is probably much easier to develop. True, AO can be problematic, but with proper timing, cell and dye concentrations, fixation, and buffers, AO staining and analysis can be very straightforward. Users also express concern over cytometer contamination with residual AO . . . we have no problem with this. Simply be diligent about flushing your sample system with 10% bleach after AO. I've found that with this, AO cross-contamination is a non-issue. If you're interested, I'll try to outline our AO staining protocol, though I'm sure there's plenty in the literature to direct you. MAK. Jeff Louie wrote: > I am trying to locate a flow cytometry laboratory in the Los Angeles area > that can provide differential staining of DNA and RNA on cell culture > material using dyes such as acridine orange (AO) or a combination of pyronin > Y (PY) and Hoechst 33342 (PY-Hoeschst 33342). Our laboratory has a MoFlo with > appropriate lasers (argon and UV) to analyse this combination of dyes but > have no experience with this particular application. The PI that wants the > work done will pay for the service but has a tight deadline (middle of > August) to complete the work. I would apprecaite very much any leads to labs > doing this work or comments as to the difficulty of setting it up in-house. > Thank you for your response.
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