From: Albert D Donnenberg, PhD (donnal@novell1.dept-med.pitt.edu)
Date: Sat Nov 23 1996 - 19:20:04 EST
Flow'ers Thanks so much for your advice on how to sharpen PI cvs when staining for DNA content and intracellular proteins. Here is a compendium of the responses: From: R.Knuechel, MD Institute of Pathology University of Regensburg <ruth.knuechel-clarke@klinik.uni-regensburg.de> First of all PI staining quality is dependent on cellular fixation, as you probably know, and fixation in methanol (cold 70% ) gives the nicest results for a lot of people including us. When nuclear antibodies are included, we have good experience with a paraformaldehyde/Tween protocol For your nuclear antibodies, you probably stain after you have prepared nuclear suspensions? The quality of this also effects the CV, and also, although it may be trivial the cell number you have availabel for your analysis. From: jim phillips univ. of miami jphillip@mednet.med.miami.edu Check your fix. ethanol at 70% in water for a min. of 18 hrs. Put the pi into the sample only 20 min to an hour before you run the sample. Make sure the tip and the machine are clean, run some 20% bleach through the system to clean the nozzle and get rid of protein. run your sample on low or at a slow rate. do some live gating on width and area plots. this gets rid of the doublets. I hope that this helps some. From: Lisa Adams LAADAMS@am.pnu.com It may be dependent on cell type and/or your fixation protocol. I have found there is a trade-off between good antibody binding and good DNA histograms when I use paraformaldehyde as a fixative (which is what works best in my hands). It has been my experience that when I start doing 2- or 3-color analysis, I can't have my cake and eat it too. From: "Martin, Jill V." <martin.jill@mayo.edu> (Jill Martin) I am sure that you have checked for doublets, and that you have a low flow rate, but these would be two sources of CV error. From: tom_frey@bdis.com You don't mention whether you are using an ethanol technique or one of the now-more-common PFA/detergent methods or products. Length of PFA fixation and length of time in PFA free buffer after staining can both alter PI CVs. See Cytometry 12:279 for example. ******************************* Albert D. Donnenberg, PhD Associate Professor of Medicine University of Pittsburgh W1056 BST 200 Lothrop St. Pittsburgh, PA 15213 (412) 624-9596 *******************************
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