This protocol was designed to be used with live cells, although I have used it with a wide variety of animal and plant cells that have been fixed. With live cell suspensions, nuclei are freed by hypotonic lysis. RNase does need to be added as PI will label double stranded RNA as well as double stranded DNA. In the abscence of RNase, peak resolution is less precise. (RNase is added at 30 Kuenitz units/mL.) In the technique described by Tate et a1. (see Cytometry 4:211-215, 1983 for detailed protocol), NaCl is added to gain normal tonicity before analysis as Na increases PI fluorescence intensity. Dave ======================================== David M. Coder, Ph.D. Consultant in Cytometry email: d_coder@msn.com or dcoder1@hotmail.com tel./messages: 206-499-3446 ----- Original Message ----- From: "Yalin Guo" <guo@mm11.ukl.uni-freiburg.de> To: cyto-inbox Sent: Tuesday, March 26, 2002 6:12 AM Subject: a question on cell cycle analysis with PI > > I am doing a cell cycle analysis using cultured and non-cultured > human CD34+ cells. I got a protocol with PI staining (0.1% Na > Citrate, 0.1% Triton X-100, 20 ug/ml PI), but without fixation > and RNase. I should be very grateful to get any suggestions for > this method. > > Yalin > >
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