Dear Kerstin To clarify a few points about live and dead. Permeabilized does not necessarily mean dead as there is plenty of literature telling you about transient permeabilisation. However, if you can exclude that it's the closest you get to dead cell detection. The use of solvents and detergents can also alter dye exclusion properties or add to toxicity. Concentration will change your staining kinetics and Howard has already mentioned the problems of active dye transport in either way that will add to that effect. At low dye concentration you will also experience more signal variation based on the amount of binding sites available to the dye. Thus the amount of free double stranded RNA will reduce the amount of free PI and your histograms will change with your cell numbers. The way you set your regions is another factor contributing to variation in % positive cells, particular if you include weak positive cells. Perhaps you can put some example data on your web site for further discussion Happy Easter Gerhard -----Original Message----- From: Kerstin Büttner [SMTP:k.buettner@uke.uni-hamburg.de] Sent: Wednesday, April 04, 2001 12:14 PM To: Cytometry Mailing List Subject: Viability PI vs. AAD - concentration and compensation 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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