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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