Ron Rabin writes: >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. > If you have no problems resolving Red613 from PE and PECy5, doing the 4-color immunofluorescence in the 488 beam and Hoechst 33342 in the UV beam is probably preferable to trying to get a good DNA histogram with 7-AAD. Whether or not you can get good staining without fixing or permeabilizing the cells depends on cell type; incubation at 37 C with 3-5 ug/ml Hoechst 33342 for 30 minutes or so generally gives good staining of viable human or rat lymphoid cells, but many strains of mice seem to pump out the dye, and some hematopoietic cells, e.g., the more primitive CD34+ cells, from many mammalian species are Hoechst-dim. At 1 ug/ml, Hoechst 33342 gives good DNA histograms in fixed cells, particularly when 0.1% Triton X-100 is added. However, I (and others) have observed, but not formally published, that staining of formaldehyde-fixed cells with Hoechst dyes and DAPI produces increases in fluorescence background in the fluorescein and PE channels. We don't understand the mechanism, but believe that the fluorescence is due to a chemical reaction between the dye and formaldehyde or some product of formaldehyde. The longer the time in fixative, the worse the background; once cells have been adequately fixed, adding 0.15% glycine (a suggestion made to us by Richard Riese) seems to neutralize the remaining fix and prevent further increases in background fluorescence. If I weren't trying to use the formaldehyde to neutralize HIV and other nasties in specimens, I'd be tempted to work with unfixed or ethanol-fixed cells. -Howard
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:31 EST