We are doing cell cycle analysis on a BD LSR with Hoechst and Pyronin Y. The ultimate goal is to look at cells that are tetramer positive. The tetramer is APC conjugated, and we are also using FITC CD8. The problem is that a small percentage (about 0.3%) of cells are "spilling" into the tetramer positive APC gate in the absence of tetramer and only in the presence of pyronin Y. The level of Pyronin staining does correlate with the spillover, but if we titer the Pyronin, we have compensation problems with the FITC. Since the percentage of tetramer positive cells is also about 0.3-1%, the percentage of spurious tetramer positive cells in the gate is significant. Does anyone know why this is a problem? What is very odd is that this appears to be unique to the LSR. When the cells are analyzed on a FACSCalibur, this spillover doesn't occur. Unfortunately, we need to use Hoechst as our DNA stain. thanks. ron Ronald L. Rabin, M.D. Senior Staff Fellow Laboratory of Immunobiochemistry DBPAP/OVRR Center for Biologics Evaluation and Research U.S. Food and Drug Administration 29 Lincoln Drive (MSC-4555) Building 29, Room 129 Bethesda, MD 20892-4555 phone: 301.496.8806 fax: 301.402.5177 email: rr84g@nih.gov
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