From: Christopher J. Groves (CGroves@infonet.tufts.edu)
Date: Thu Dec 12 1996 - 09:04:54 EST
Dr. Ormerod, I have noticed the same problem with PBL's and sometimes with activated lymphs and cell lines, with PI, DAPI, 7-AAD, and Ho258 as well. I usually gate based on an activation or other cell specific protein marker. I have a feeling that what is seen is that the DNA packing (for lack of a better word) and coiling is different with differing cell types and during activation. What might be observed is that when you use a detergent step, you break down the histones. Typically an acid treatment will work to break these down as well, I use citric acid trisodium salt with my DAPI. You can easily tell by adding some acid to a fraction of your PI and see if there's a difference. The cloud to this silver lining may be that if you want to use TdT, which is typically conjugated to FITC, the acid will render the FITC useless due to it's pH sensitivity. I find it difficult to believe that the different cells have differing amounts of DNA, let me know if I'm wrong. I'm curious as to what the actual cause would be. I'd appreciate it if you posted your conclusion. Best of luck. Cheers, Chris Groves Tufts University School of Medicine Boston, MA USA
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