Ronald Rabin wrote: > I am planning to do 4 color analysis + cell cycle on a dual laser Vantage that > can use either a dye laser or a UV laser as the second laser. I can use > either FITC, PE, PECy5, Red613 and Hoechst, or I can use FITC, PE, 7ADD, R613, > and APC. Because my PE conjugates are critical to the experiment, I cannot > use the Pyronin Y. It seems the Hoechst would be easier, cell permeable etc, > though I have no experience with it. Ron,If cell cycle distribution is your goal, then Hoechst 33342 would be the way. Although somewhat sensitive to chromatin conformational differences, it will provide a reasonable indication of relative DNA content. An added advantage is not having to kill (permeabilize) the cells before you stain them. Staining requires optimization re. concentration, time, etc.. Also, you will have to incubate at 37C, which can cause problems with your surface staining . . . something to consider. Hoechst, however, will also work very nicely on aldehyde fixed cells (be sure to reduce the dye concentration to about 1/10 used for live cells), so this may not be a factor. If you do decide to go Hoechst 33342 in fixed cells, you might consider DAPI as alternative UV excitable DNA stain. Much less sensitive to conformation -- probably the best in that regard. 7AAD can be used as well, but there are problems with stoichiometry and different cell types. I've used it to look at the kinetics of T cell repopulation, so it works, but 7AAD is very sensitive to time/concentration/temperature, often prohibitively so. If the only reason you go with it is to change the color, there are other dyes that may work (Molecular Probes), but you may have to figure them out. All things considered, Hoechst or DAPI are your best bet. If what you care about is a viability marker . . . that's a different discussion. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuru@umich.edu
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:30 EST