Kerstin, Dead cells are stained with PI almost immediately. There's really no reason to wait several minutes. Also, RNAse is added when we stain for DNA content analysis . . . it improves resolution . . . and is not necessary in a "live/dead" application. I haven't actually looked at it, but I also believe 7-AAD staining of DNA in dead cells will be "immediate." I'm not surprised there's a concentration dependant difference, although I think you'll see this more with 7-AAD than with PI (since PI is so much brighter). I've gone as low as 0.5 ug/ml PI and still seen good dead cell detection, but also know that others will recommend higher dye concentrations . . . Regarding your "compensate/no compensate" question . . . I think this depends somewhat on what you use the PI for. If you're interested in detecting live vs dead cells, I think it's important to correctly compensate so that you can determine live/dead cutoffs amongst all parameters. This is easier to achieve at lower dye concentrations, since the amount of crossover will increase with signal intensity. In a dead cell exclusion scenario, compensation is irrelevant since the PI positive cells are excluded by gating. We usually compensate these anyway . . . again because it looks better . . . but it's really not necessary. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Flow Core 7416 CCGC 0946 (734) 647-3216, fax (734) 936-7376 kukuru@umich.edu >>> Kerstin Büttner <k.buettner@uke.uni-hamburg.de> 04/04/01 07:14AM >>> Hello flowers, I am stuck with a very basic problem. I am trying to separate dead from viable cells. People in my lab have always done this with 10µgPI/ml plus RNase for 20min room temperature. I have tried two other protocols: PI 2µg/ml 5min on ice and AAD 1µg/ml 30min on ice. Every protocol yields different counts of dead cells - from 16 +/-3% (AAD), 22+/-7% (PI) to 27+/-5% (PI combined with RNase). Is this a probem of concentration - are DNA-Dyes titreable like an antibody? Is it a question of temperature or time? We do not understand this, because we always thought that a dead cell is dead, has permeabilised membranes and therfore takes up the dye - not depending on concentration. We always thought that higher concentration only gives you a better separation of peaks. How do I know which concentration is the right one for my AAD? And there ist still another problem with PI: If I stay with PI - is it more accurate to compensate PI out of FL2 (which in fact means 99,9% compensation) or is it better to leave the PI signal where it is, since the dead cells are gated out anyway before the PE stained cells in FL2 are analyzed? Thanks very much for any comments on these issues! Kerstin Kerstin Buettner Institute of Biophysics and Radiobiology University of Hamburg Martinistr. 52 20246 Hamburg Phone: 0049-40-42803-2938 Fax: 0049-40-42803-5139 E-Mail: k.buettner@uke.uni-hamburg.de
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