Dear Mark, Thank you for your recommendations, I will take them into account. I was following the reference: Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis.In: Methods in cell biology, Volume 41 Flow Cytometry, 2nd edition, part A. 1994. Lars L. Vindelov and Ib Jarle Christensen. I will try with lower concentrations of IP in order to improve my CVs (now about 4% for solid tissues). Ana Mark Kukuruga escribió: > Ana, > I think you should re-check that . . . every reference I've looked at for Vindelov's (as I just did . . .) indicates 50 ug/ml as the PI concentration. > Also, I've analyzed cells stained at PI concentrations above 50 . . . the PI signal starts to drop at about 80 ug/ml, and %c.v. increases dramatically. > Finally . . . we use PI to exclude dead cells in extended sorts . . . little toxic effect is evident. > MAK. > > -- > Mark A. KuKuruga, Managing Director > University of Michigan Flow Core > 7416 CCGC 0946 > (734) 647-3216, fax (734) 936-7376 > kukuru@umich.edu > > >>> Ana Salas <ana@spi.uniovi.es> 06/22/01 05:08AM >>> > Hello everbody, > When you are measuring cell cycle with the intercalating dye IP you must be sure that the > concentration is in great excess to saturate all nuclei. Then, you should wait for 10 > minutes to obtain an equilibrium between IP inside and the solution and then you can make > the adquisition. If cell concentration is too high is not possible to became stoichiometric > join IP-DNA. In Vindelov protocol IP concentration (for uniparametric analysis) is about 140 > ug/ml for 1 million of cells. > When you want to measure cell viability with IP the concentration of cells and time is also > important. The concentration should be under 1ug/ml (for 1 million of cells) and the > adquisition should be make inmediately to avoid cells to die due to the toxic effect of the > IP. > Chao > > Ana Salas > Servicio científico-técnico de Citometria > Universidad de Oviedo.Spain > > Mark Kukuruga escribió: > > > 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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