Ronald, I have been running Hoechst, Pyronin Y, and APC on my LSR and I have not seen the background in the APC channel that you suggest. In fact, I am seeing literally no background in the APC channel. I suppose that you could have a problem with compensation as I have seen effects on the APC channel while adjusting, for example, FL3-FL1 or FL2-FL1. Another possibility is the quality of your Pyronin Y. You should be using Pyronin Y from Polysciences as this is very pure. You could also have a problem with the dichroic configuration as you will find that with small adjustments of dichroic angle you will see changes in background levels in various channels. The last thing to check is that all lasers are accurately hitting the pinholes in front of PMT filters. David David M. Dombkowski dombkowski@helix.mgh.harvard.edu Flow Cytometry-Pathology-CNY rm7017 149 13th Street Massachusetts General Hospital-East Charlestown, MA 02129 Tel. (617)726-1683 Fax.(617)724-3164
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