Philippe, First . . . as a marker for viability, calcein AM (Molecular Probes C-1430) is a better dye than FDA, due to its better retention in cells and relative insensitivity to pH. You can read about this (and other viability probes) at the Molecular Probes site . . . ://www.probes.com/handbook/sections/1502.html> Second . . . related point . . . it's always good to measure membrane intactness simultaneous with a viability probe. Adding PI or 7-AAD will improve your live/dead discrimination, and may help to detect artifacts due to inadequate staining or dye efflux. Finally . . . to answer your question . . . thymocytes are considerably smaller, and therefore may carry far less dye. Most likely, the signal from your 293 cells is too bright at the concentration you're using to be effectively managed compensation-wise. Look at lower concentrations of your viability dye. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Philippe Pognonec <Philippe.Pognonec@unice.fr> 11/26/01 03:58AM >>> Hi there. We are using FDA as a marker of living/dead cells in thymocytes preparations on a FACSort, in FL1. We do not observe any uncontrolable leakage of the flurescence in FL2, and are pretty happy with it. Fine. No we try the same stuff on 293 cells (after dissociation following light trypsination), and we get teh same signal in FL1, 2 and 3!!!! Impossible to use any other marker at the same time. Does anybody have any idea what's wrong? Philippe ____________________________________________ Philippe Pognonec, Ph.D. Transcriptional Regulation and Differentiation Centre de Biochimie Parc Valrose Universite de Nice 06108 Nice cedex 2 France Tel/fax: (33) 492 07 64 13 http://www.unice.fr/biochimie/u470/prog5us.htm ____________________________________________
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