Rana, PI stains double-stranded nucleic acids. If we want to resolve cell cycle compartments, we typically use 20-50 ug/ml (I've gone as low as 10) as a staining concentration. If you do the comparison, you'll find that this relatively broad range will yield comparable results. Much higher, and you start to lose resolution . . . lower, and you may no longer be stoichiometric . . . i.e., you may not stain all cells as uniformly as you'd like, and G1/G2M ratios will suffer. 50 ug/ml is an accepted concentration for cell cycle staining. However, if all you need is a viability marker, add just enough to resolve live from dead. And, since this measurement is usually done log amplified, only a little is needed. There are applications where sub-optimal PI concentrations are necessary. If, for example, you want to look at other nucleic acid-associated markers (say, incorporated BrdU, or some DNA associated protein) with green fluorescent dyes - - like FITC - - , you may actually quench your signal with too much PI. Here, stoichiometry is sacrificed for green signal resolution . . . in this case, all you want is a G1 and G2M position indicator, and 1 to 5 ug/ml PI will accomplish that. Adding lower amounts of PI will better allow simultaneous analysis of phycoerythrin conjugates . . . signal crossover is far more manageable. Adding low amounts also helps with visualizing co-stains via microscopy. If, for example, you want to look at PI versus some green fluorescent marker . . . too much PI will overwhelm/mask the green signal. Bottom line . . . use the concentration that's appropriate for the application. 50 ug/ml is a standard for cell cycle. Other applications may require changes in this empirically defined optimal concentration. Using PI at 1 ug/ml (or even less) will work as a viability indicator. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Rana Nagarkatti <rana_nagarkatti@usa.net> 06/19/01 05:06PM >>> Dear Group, I was wondering if the above queston is actually true. The reason being that I had done some calculations regarding the final conc of PI in various protocols used for Viability and DNA analysis. and found that ususally for DNA 50ug/ml is the required conc ( for Flow- may be to maintain equilibrium in the stream etc but any ideas for fluorimetry???)and for viability it is around 1ug/ml. What is the reason? Do higher conc of PI in the staining buffer mean that more PI will enter the cells or is the cell totally impermeable to the PI. or that the PI conc used for viability has been standardized based on a concurrent experiment with say trypan blue or other methods? And how does time of incubation after adding the dye relate to the conc. After all both the methods are based on the same prinnciple that PI binds to DNA. Are there any simple proportionalities involved here eg more the dye less the time or mmore the dye more enters into the cells etc. Thanks in advance for all the tips. Rana Nagarkatti ____________________________________________________________________ Get free email and a permanent address at http://www.netaddress.com/?N=1
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