Ann PI binding to DNA is an equilibrium binding reaction. Thus, changing the concentration of either reactant will change the concentration of product (the PI/DNA complex and thus the fluorescence). You will notice this particularly if you stain for very short periods of time - the binding has not reached equilibrium and potentially 2 cells with exactly the same amount of DNA will have a slightly different fluorescence because the cells have bound different amounts of PI due to having experienced different kinetics in binding - different local concentrations of either reactant, slight cell permeability differences etc.. Giving the reaction sufficient time will result in these 2 cells having the same fluorescence. You notice this effect as less than optimal CVs. If you have cell/PI concentrations that change but are at saturation for the PI these samples should end up with the same fluorescence given sufficient time to reach equilibrium. However, if PI is not saturating then even if you give enough time to reach equilibrium (ie. all cells with the same DNA content have bound the same amount of PI) the fluorescence of each cell will be less than when using PI at saturation since there is less product (PI/DNA complex). This is what you see as the G1 cells ,for example, being in a different channel position from one sample to the other. Using a sub-saturating concentration of PI will exacerbate the first problem since local concentrations of reactants can vary more and CVs will be worse. Reaching equilibrium at the non-saturating concentration will give good CVs even though the per cell fluorescence is less. Larry At 03:34 PM 12/18/2000 +0100, you wrote: >Hallo everybody, > >The users here are encouraged to follow protocols from "Current Protocols >in Cytometry" whenever possible. Recently we've started doing a lot of DNA >staining and have noticed variation in fluorescence between similar samples >when utilising PI. On scrutinising the given protocol in CPC, it says you >can have a cell suspension of 1 million to 10 million cells for PI. For >Dapi it says 100 thousand to 1 million, and for Hoechst a suspension of 1 >million. Somewhere in the past I learnt that you use a constant number of >cells depending on the protocol. > >I take it it should mean you can stain up to a certain concentration of >cells but omits to explain that the cell number per sample should not vary. > >However, if you can vary the cell concentration to such an extent surely >the A-T bases are no longer saturated and will give sub-optimal staining. >Oui??? > >In the case of Hoeschst where the users go by the 1 million cell conc. we >do not see the peaks shifting up and down between samples. > >Regards >Ann > >Merry Xmas Larry W. Arnold, Ph.D. Res. Assoc. Prof. Director, Flow Cytometry Facility Department of Microbiology and Immunology Lineberger Comprehensive Cancer Center CB# 7290 University of North Carolina at Chapel Hill Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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